Synthesis of aryl azide derivatives of UDP-GlcNAc and UDP-GalNAc and their use for the affinity labeling of glycosyltransferases and the UDP-HexNAc pyrophosphorylase.

The chemical synthesis and utilization of two photoaffinity analogs, 125I-labeled 5-[3-(p-azidosalicylamido)-1-propenyl]-UDP-GlcNAc and -UDP-GalNAc, is described. Starting with either UDP-GlcNAc or UDP-GalNAc, the synthesis involved the preparation of the 5-mercuri-UDP-HexNAc and then attachment of an allylamine to the 5 position to give 5-(3-amino)allyl-UDP-HexNAc. This was followed by acylation with N-hydroxysuccinimide p-aminosalicylic acid to form the final product, i.e., 5-[3-(p-azidosalicylamido)-1-propenyl]-UDP-GlcNAc or UDP-GalNAc. These products could then be iodinated with chloramine T to give the 125I-derivatives. Both the UDP-GlcNAc and the UDP-GalNAc derivatives reacted in a concentration-dependent manner with a highly purified UDP-HexNAc pyrophosphorylase, and both specifically labeled the subunit(s) of this protein. The labeling of the protein by the UDP-GlcNAc derivative was inhibited in dose-dependent fashion by either unlabeled UDP-GlcNAc or unlabeled UDP-GalNAc. Likewise, labeling with the UDP-GalNAc probe was blocked by either UDP-GlcNAc or UDP-GalNAc. The UDP-GlcNAc probe also specifically labeled a partially purified preparation of GlcNAc transferase I.

Purification to homogeneity and properties of UDP-GlcNAc (GalNAc) pyrophosphorylase.

The pyrophosphorylase that condenses UTP and GlcNAc-1-P was purified 9500-fold to near homogeneity from the soluble fraction of pig liver extracts. At the final stage of purification, the enzyme was quite stable and could be kept for at least 4 months in the freezer with only slight loss of activity. On native gels, the purified enzyme showed a single protein band, and this band was estimated to have a molecular mass of approximately125 kDa on Sephacryl S-300. SDS-polyacrylamide gel electrophoresis analysis of the enzyme gave three protein bands of 64, 57, and 49 kDa, but these polypeptides are all closely related based on the following. 1) All three polypeptides show strong cross-reactivity with antibody prepared against the 64-kDa band. 2) All three proteins become labeled with either the UDP-GlcNAc photoaffinity probe azido-125I-salicylate-allylamine-UDP-GlcNAc or a similar UDP-GalNAc photoaffinity probe, and either labeling was inhibited in a specific and concentration-dependent manner by unlabeled UDP-GlcNAc or UDP-GalNAc. Thus, the enzyme is probably a homodimer composed of two 64-kDa subunits. The purified enzyme had an unusual specificity in that, at higher substrate concentrations, it utilized UDP-GalNAc as a substrate as well as UDP-GlcNAc in the reverse direction and GalNAc-1-P as well as GlcNAc-1-P in the forward direction. However, the Km for the GalNAc substrates was considerably higher than that for GlcNAc derivatives. This activity for synthesizing UDP-GalNAc was not due to epimerase activity since no UDP-GalNAc could be detected when the enzyme was incubated with UDP-GlcNAc for various periods of time. The pyrophosphorylase required a divalent cation, with Mn2+ being best at 0.5-1 mM, and the pH optimum was between 8.5 and 8.9.

Synthesis of 5-IASA-UDP-GlcNAc and its use for the photoaffinity labeling of a novel UDP-GlcNAc pyrophosphorylase.

Photoreactivable 5-[3-(p-iodoazidosalicylamide)allyl]-UDP-GlcNAc (5-IASA-UDP-GlcNAc) was synthesized by a four-step procedure and used for photoaffinity labeling of UDP-GlcNAc-dependent enzymes. Upon iodination with 125I, the compound was successfully applied to probe a purified UDP-GlcNAc pyrophosphorylase from pig liver. The enzyme was photoinactivated by the probe in the concentration-dependent manner, and was protected by UDP-GlcNAc and, to a lesser extent, by UTP and UDP-GlcCOOH.

GDP-mannose pyrophosphorylase. Purification to homogeneity, properties, and utilization to prepare photoaffinity analogs.

Pig liver GDP-mannose pyrophosphorylase was purified 5,000-fold to apparent homogeneity using standard techniques. The native enzyme showed a single band on gels of about 450 kDa and two subunits of 43 and 37 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 37-kDa (beta-) subunit had only methionine at its amino terminus and a surprisingly hydrophobic sequence: Met-Lys-Ala-Leu-Ile-Leu-Val-Gly-Gly-Tyr-Gly-Thr-Arg-Leu- Arg-Pro-Leu-Thr-Leu-Ser-Ile-Pro-Lys. The 43-kDa (alpha-) subunit was blocked at the amino terminus, but a 29-kDa CNBr fragment had the following sequence: Leu-Asp-Ala-His-Arg-His-Arg-Pro-His-Pro- Phe-Leu-Leu-. Substrate specificity studies done in the direction of formation of nucleoside triphosphate and sugar-1-P indicated that the enzyme was most effective with GDP-glucose as substrate (100%) followed by IDP-mannose (72%) and then GDP-mannose (61%). That GDP-mannose and GDP-glucose activities were indeed catalyzed by the same enzyme was indicated by the following. (i) Various studies indicated that the enzyme was homogeneous. (ii) A staining procedure for production of GTP stained the same single band on native gels when either GDP-mannose or GDP-glucose was the substrate. (iii). GDP-mannose inhibited the utilization of GDP-glucose by the enzyme, and vice versa. When 8-azido-[32P]GTP was incubated with native enzyme and exposed to UV light, both the 43-kDa and the 37-kDa subunits became labeled, although the 37-kDa subunit reacted more strongly. On the other hand, 8-azido-GDP-[32P]mannose only photolabeled the 43-kDa band. Most importantly, the purified enzyme can be utilized to produce 8-azido-[32P]GDP mannose or 8-azido-[32P]GDP glucose.

Immobilized metal ion affinity chromatography in enzyme fractionation.

Ribitol dehydrogenase from Mycobacterium butyricum and alpha-mannosidase from Lupinus luteus seedlings were fractionated by the immobilized metal ion (Cu2+ or Zn2+) affinity chromatography (IMAC) on iminodiacetic acid coupled to Sepharose 6B. In a single step, ribitol dehydrogenase was purified 10-12 fold with the recovery above 80% when using Zn(2+)-Sepharose 6B as the sorbent and decreasing linear gradient of pH from 7 to 4. In the same conditions purification of alpha-mannosidase was less effective (2-3 fold, recovery 60-70%).

Purification to homogeneity and properties of mannosidase II from mung bean seedlings.

Mannosidase II was purified from mung bean seedlings to apparent homogeneity by using a combination of techniques including DEAE-cellulose and hydroxyapatite chromatography, gel filtration, lectin affinity chromatography, and preparative gel electrophoresis. The release of radioactive mannose from GlcNAc[3H]Man5GlcNAc was linear with time and protein concentration with the purified protein, did not show any metal ion requirement, and had a pH optimum of 6.0. The purified enzyme showed a single band on SDS gels that migrated with the Mr 125K standard. The enzyme was very active on GlcNAcMan5GlcNAc but had no activity toward Man5GlcNAc, Man9GlcNAc, Glc3Man9GlcNAc, or other high-mannose oligosaccharides. It did show slight activity toward Man3GlcNAc. The first product of the reaction of enzyme with GlcNAcMan5GlcNAc, i.e., GlcNAcMan4GlcNAc, was isolated by gel filtration and subjected to digestion with endoglucosaminidase H to determine which mannose residue had been removed. This GlcNAcMan4GlcNAc was about 60% susceptible to Endo H indicating that the mannosidase II preferred to remove the alpha 1,6-linked mannose first, but 40% of the time removed the alpha 1,3-linked mannose first. The final product of the reaction, GlcNAcMan3GlcNAc, was characterized by gel filtration and various enzymatic digestions. Mannosidase II was very strongly inhibited by swainsonine and less strongly by 1,4-dideoxy-1,4-imino-D-mannitol. It was not inhibited by deoxymannojirimycin.

The hypoglycaemic response of diabetic rats to insulin-liposomes.

We prepared insulin-liposomes using one combination of lipids including phosphatidylcholine (cholesterol) stearylamine, 7/2/1 (molar ratio). Non-sonicated liposomes (LMV) and sonicated liposomes (SUV) contained about 20% and 5% of insulin, respectively. Free insulin was removed from liposomes-associated insulin by ultracentrifugation, or ultrafiltration on Sepharose 6B column. Insulin preparations were administered parenterally and non-parenterally into male, Wistar rats with alloxan diabetes to produce the hypoglycaemia. In case of i.v. and s.c. routes of administration all preparations acted in the similar manner giving the clear hypoglycaemia after 2 h. When administered intragastrically only liposome insulin caused hypoglycaemia. In case of buccal and nasal routes of administration only SUV-insulin was effective.

Hypoglycemic effect of liposome-entrapped insulin administered by various routes into normal rats.

We entrapped insulin into liposomes using one combination of lipids comprising egg lecithin-cholesterol-stearylamine (7:2:1 molar ratio). The efficiency of entrapment was about 20% with unsonicated liposomes (LMV), and around 5% with sonicated liposomes (SUV). LMV-, SUV- and free-insulins were administered via different routes into male, non-diabetic Wistar rats in order to change the glycemia. When administered parenterally all preparations acted in a similar manner, reducing the glycemia after 2 h in the range of 75% to 85% in case of iv administration, and 43% to 67%--after sc administration. Only liposome insulin acted via intragastric route, and SUV-insulin-via buccal--or nasal route.

Subcellular localization of glycosidases and glycosyltransferases involved in the processing of N-linked oligosaccharides.

Using isopycnic sucrose gradients, we have ascertained the subcellular location of several enzymes involved in the processing of the N-linked oligosaccharides of glycoproteins in developing cotyledons of the common bean, Phaseolus vulgaris. All are localized in the endoplasmic reticulum (ER) or Golgi complex as determined by co-sedimentation with the ER marker, NADH-cytochrome c reductase, or the Golgi marker, glucan synthase I. Glucosidase activity, which removes glucose residues from Glc(3)Man(9)(GlcNAc)(2), was found exclusively in the ER. All other processing enzymes, which act subsequent to the glucose trimming steps, are associated with the Golgi. These include mannosidase I (removes 1-2 mannose residues from Man(6-9)[GlcNAc](2)), mannosidase II (removes mannose residues from GlcNAcMan(5)[GlcNAc](2)), and fucosyltransferase (transfers a fucose residue to the Asn-linked GlcNAc of appropriate glycans). We have previously reported the localization of two other glycan modifying enzymes (GlcNAc-transferase and xylosyltransferase activities) in the Golgi complex. Attempts at subfractionation of the Golgi fraction on shallow sucrose gradients yielded similar patterns of distribution for all the Golgi processing enzymes. Subfractionation on Percoll gradients resulted in two peaks of the Golgi marker enzyme inosine diphosphatase, whereas the glycan processing enzymes were all enriched in the peak of lower density. These results do not lend support to the hypothesis that N-linked oligosaccharide processing enzymes are associated with Golgi cisternae of different densities.

Purification and properties of the glycoprotein processing N-acetylglucosaminyltransferase II from plants.

The presence of an N-acetylglucosaminyltransferase (GlcNAc-transferase) capable of adding a GlcNAc residue to GlcNAcMan3GlcNAc was demonstrated in mung bean seedlings. This enzyme was purified about 3400-fold by using (diethylaminoethyl)cellulose and phosphocellulose chromatographies and chromatography on Concanavalin A-Sepharose. The transferase was assayed by following the change in the migration of the [3H]mannose-labeled GlcNAc beta 1,2Man alpha 1,3(Man alpha 1,6)Man beta 1,4GlcNAc on Bio-Gel P-4, or by incorporation of [3H]GlcNAc from UDP-[3H]GlcNAc into a neutral product, (GlcNAc)2Man3GlcNAc. Thus, the purified enzyme catalyzed the addition of a GlcNAc to that mannose linked in alpha 1,6 linkage to the beta-linked mannose. GlcNAc beta 1,2Man alpha 1,3(Man alpha 1,6)Man beta 1,4GlcNAc was an excellent acceptor while Man alpha 1,6(Man alpha 1,3)Man beta 1,4GlcNAc, Man alpha 1,6(Man alpha 1,3)Man alpha 1,6(Man alpha 1,3)Man beta 1,4GlcNAc, and Man alpha 1,6(Man apha 1,3)Man alpha 1,6[GlcNAcMan alpha 1,3]Man beta 1,4GlcNAc were not acceptors. Methylation analysis and enzymatic digestions showed that both terminal GlcNAc residues on (GlcNAc)2Man3GlcNAc were attached to the mannoses in beta 1,2 linkages. The GlcNAc transferase had an almost absolute requirement for divalent cation, with Mn2+ being best at 2-3 mM. Mn2+ could not be replaced by Mg2+ or Ca2+, but Cd2+ showed some activity. The enzyme was also markedly stimulated by the presence of detergent and showed optimum activity at 0.15% Triton X-100. The Km for UDP-GlcNAc was found to be 18 microM and that for GlcNAcMan3GlcNAc about 16 microM.

Effect of isomers of swainsonine on glycosidase activity and glycoprotein processing.

The chemical synthesis of swainsonine [(1S,2R,8R,8 alpha R)-trihydroxyindolizidine] from trans-1,4-dichloro-2-butene was previously described [Adams, C. E., Walker, F. J., & Sharpless, K. B. (1985) J. Org. Chem. 50, 420-424]. A modification of that synthesis provided two other isomers, referred to here as "Glc-swainsonine" [(1S,2S,8R,8 alpha R)-trihydroxyindolizidine] and "Ido-swainsonine" [(1S,2S,8S,8 alpha R)-trihydroxyindolizidine]. To determine whether these new compounds had biological activity, they were compared to swainsonine as inhibitors of a number of commercially available glycosidases. While swainsonine is a potent inhibitor of jack bean alpha-mannosidase but does not inhibit other glycosidases, its two isomers were inactive on alpha-mannosidase but did inhibit other enzymes. Thus, Glc-swainsonine was an inhibitor of the fungal alpha-glucosidase amyloglucosidase, and this inhibition was of a competitive nature (Ki = 5 X 10(-5) M) with respect to the substrate p-nitrophenyl alpha-D-glucopyranoside. This alkaloid also inhibited beta-glucosidase, but much less effectively than alpha-glucosidase. On the other hand, Ido-swainsonine was more effective toward beta-glucosidase than toward alpha-glucosidase, and this inhibition was also of a competitive nature. None of these inhibitors were effective against beta-mannosidase or alpha- or beta-galactosidase. Glc-swainsonine was also tested against the glycoprotein processing glycosidases. Surprisingly, in this respect, the alkaloid was like swainsonine in that it inhibited mannosidase II but had no effect or only slight effect on glucosidase I, glucosidase II, and mannosidase I. Glc-swainsonine also inhibited glycoprotein processing in cell culture.(ABSTRACT TRUNCATED AT 250 WORDS)

6-Epicastanospermine, a novel indolizidine alkaloid that inhibits alpha-glucosidase.

A second indolizidine alkaloid, epimeric with castanospermine, has been isolated from seeds of the Australian tree Castanospermum australe. The structure was established as 6-epicastanospermine by proton and carbon-13 nuclear magnetic resonance spectroscopy and mass spectrometry. 6-Epicastanospermine was found to be a potent inhibitor of amyloglucosidase, (an exo-1,4-alpha-glucosidase), a weak inhibitor of beta-galactosidase, and not to inhibit beta-glucosidase and alpha-mannosidase. These results indicate that glycosidase inhibitory activity cannot be predicted by comparison of the structure and stereochemistry with the appropriate sugars, since 6-epicastanospermine is an analog of mannose and not of glucose. The inhibition of amyloglucosidase was found to be competitive and to be more effective at higher pH values. Castanospermine and 6-epicastanospermine differed in their effect upon the mung bean processing enzymes, glucosidase I and II, in that the former is a potent inhibitor whereas the latter is a very poor inhibitor. Subtle alterations in stereochemistry of these alkaloids can therefore produce significant changes in their biological activity.

Purification and properties of glucosidase I from mung bean seedlings.

The microsomal enzyme fraction from mung bean seedlings was found to contain glucosidase activity capable of releasing [3H]glucose from the glucose-labeled Glc3Man9GlcNAc. The enzymatic activity could be released in a soluble form by treating the microsomal particles with 1.5% Triton X-100. When the solubilized enzyme fraction was chromatographed on DE-52, it was possible to resolve glucosidase I activity (measured by the release of [3H]glucose from Glc3Man9GlcNAc) from glucosidase II (measured by release of [3H]glucose from Glc2Man9GlcNAc). The glucosidase I was purified about 200-fold by chromatography on hydroxylapatite, Sephadex G-200, dextran-Sepharose, and concanavalin A-Sepharose. The purified enzyme was free of glucosidase II and aryl-glucosidase activities. Only a single glucose residue could be released from the Glc3Man9GlcNAc by this purified enzyme and the other product was the Glc2Man9GlcNAc. Furthermore, this enzyme was inhibited in a dose-dependent manner by kojibiose, an alpha-1,2-linked glucose disaccharide, but not by other alpha-linked glucose disaccharides. These data indicate that this glucosidase is a specific alpha-1,2-glucosidase. The pH optimum for the glucosidase I was about 6.3 to 6.5, and no requirements for divalent cations were observed. The enzyme was inhibited strongly by the glucosidase processing inhibitors, castanospermine and deoxynojirimycin, and less strongly by the plant pyrrolidine alkaloid, 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine. However, the enzyme was not inhibited by the mannosidase processing inhibitors, swainsonine, deoxymannojirimycin or 1,4-dideoxy-1,4-imino-D-mannitol. The stability of the enzyme under various conditions and other properties of the enzyme were determined.

Purification and Properties of a Glycoprotein Processing alpha-Mannosidase from Mung Bean Seedlings.

The microsomal fraction of mung bean seedlings contains mannosidase activities capable of hydrolyzing [(3)H]mannose from the [(3)H]Man(9)GlcNAc as well as for releasing mannose from p-nitrophenyl-alpha-d-mannopyranoside. The glycoprotein processing mannosidase was solubilized from the microsomes with 1.5% Triton X-100 and was purified 130-fold by conventional methods and also by affinity chromatography on mannan-Sepharose and mannosamine-Sepharose. The final enzyme preparation contained a trace of aryl-mannosidase, but this activity was inhibited by swainsonine whereas the processing enzyme was not. The pH optimum for the processing enzyme was 5.5 to 6.0, and activity was optimum in the presence of 0.1% Triton X-100. The enzyme was inhibited by ethylenediaminetetraacetate while Ca(2+) was the most effective cation for reversing this inhibition. Mn(2+) was considerably less effective than Ca(2+) and Mg(2+) was without effect. The processing mannosidase was inhibited by alpha1,2- and alpha1,3-linked mannose oligosaccharides (50% inhibition at 3 millimolar), whereas free mannose and alpha1,6-linked mannose oligosaccharides were ineffective. Mannosamine was also an inhibitor of this enzyme. The aryl-mannosidase and the processing mannosidase could also be distinguished by their susceptibility to various processing inhibitors. The aryl-mannosidase was inhibited by swainsonine and 1,4-dideoxy-1,4-imino-d-mannitol but not by deoxymannojirimycin or other inhibitors, while the processing mannosidase was only inhibited by deoxymannojirimycin. The processing mannosidase was incubated for long periods with [(3)H]Man(9)GlcNAc and the products were identified by gel filtration. Even after a 24 hour incubation, the only two radioactive products were Man(5)GlcNAc and free mannose. Thus, this enzyme appears to be similar to the animal processing enzyme, mannosidase I, and is apparently a specific alpha1,2-mannosidase.

Demonstration of GlcNAc transferase I in plants.

A solubilized enzyme preparation from mung bean seedlings catalyzed the transfer of GlcNAc from UDP-GlcNAc to the Man5GlcNAc acceptor to form GlcNAc-Man5GlcNAc. In the presence of the mannosidase inhibitor, swainsonine, this oligosaccharide accumulated, but in the absence of this inhibitor, the oligosaccharide was processed further to smaller sized oligosaccharides with the release of radioactive mannose. The formation of GlcNAc-Man5GlcNAc required the presence of Man5GlcNAc, UDP-GlcNAc, Mn++ and swainsonine. The product, GlcNAc-Man5GlcNAc was characterized by chromatography on calibrated columns of Biogel P-4, and by various enzymatic digestions. These data indicate the presence of GlcNAc transferase I and mannosidase II in plants.

A simple and reliable assay for glycoprotein-processing glycosidases.

A simple and reproducible assay to measure the activity of the glycoprotein-processing glycosidases, i.e., glucosidases and mannosidases, is described. This assay takes advantage of the fact that high-mannose and glucose-containing high-mannose oligosaccharides bind to columns of concanavalin A-Sepharose, but the liberated glucose and mannose residues emerge from these columns in the wash. Thus, using [3H]mannose-labeled Man9-N-acetylglucosamine (Man9GlcNAc) or [3H]glucose-labeled Glc3Man7-9-GlcNAc as substrates, the amount of radioactivity in the wash can be quickly and efficiently determined as a measure of enzyme activity. Although the use of this assay was reported previously [B. Saunier et al., 1982, J. Biol. Chem. 257, 14155-14161], the details of its use, its reproducibility, and the problems with interfering materials have not been thoroughly described. In this report, we show that the assay is linear with time and protein concentration, and shows the expected kinetics with various processing inhibitors. The assay works well with the microsomal enzyme preparation and with a solubilized enzyme fraction. In addition, methods are described for the preparation of various radioactive oligosaccharide substrates (i.e., Man9GlcNAc and Glc3Man7-9GlcNAc) using appropriate glycoprotein-processing inhibitors.

Pharmacokinetics of prethcamide following rapid intravenous injection and oral administration in rabbits.

A simple gas chromatographic method for the quantitative determination of ethyl butamide and propyl butamide, the active constituents of the analeptic drug "Prethcamide", in whole blood and tissues has been developed. The method was used to study the disposition of the compounds after iv and po administration to rabbits. The course of changes of ethyl butamide and propyl butamide concentration following rapid intravenous injection and oral administration was described by the two-compartment and one-compartment open models, respectively. The mean half-life (t1/2 beta, min), the body clearance (Clb, 1/min), the mean absorption rate constant (ka, min1-) and tmax (min) values were, respectively: 43.07 +/- 10.54, 0.05 +/- 0.0125, 0.033 +/- 0.0073 and 30.0 +/- 5.0 for ethyl butamide and 30.0 +/- 4.13, 0.0962 +/- 0.0269, 0.0512 +/- 0.0328 and 30.0 +/- 5.0 for propyl butamide. The low bioavailability (F) of the compounds, in the range 24-32%, may be attributed to low absorption of the drugs from the gastro-intestinal tract, or the first-pass effect, or both. The drugs accumulate predominantly in the liver. The faster metabolism and elimination of propyl butamide is postulated.

A study on the Prethcamide hydroxylation system in rat hepatic microsomes.

Ethyl butamide and propyl butamide, the active constituents of the analeptic drug named Prethcamide (Ciba-Geigy), undergo biotransformation to respective single metabolites in the presence of rat hepatic microsomes and the NADPH-generating system. Spectral analysis showed that the metabolites were hydroxylated forms of the drug. The hydroxylation was stimulated by NADH and increased ionic strength, and inhibited by the known cytochrome P-450 inhibitors, e.g. SKF-525A, metyrapone, CO and KCN. The drug formed type I binding spectrum with cytochrome P-450.

The possible occurrence of zinc in ribitol and sorbitol dehydrogenases from Mycobacterium sp. 279.

The activity of polyhydric alcohol dehydrogenases in Mycobacterium sp. 279 was studied under limitation of zinc in the growth medium. It was found that the activity of ribitol and sorbitol dehydrogenases were markedly lowered and that of D-arabinitol dehydrogenase remained unchanged in the Zn2+-deficient cells. Other ions tested i.e., Co2+, Cu2+, Ni2+ and Mn2+ failed to substitute Zn2+ ions in their effect on the enzyme activities. The Zn2+-responsive enzymes were sensitive to the chelating agents (1,10-phenanthroline, 2,2'-dipyridyl), whereas D-arabinitol dehydrogenase was insensitive. The results indicate possible existence of a zinc component in the ribitol and sorbitol dehydrogenases from Mycobacterium sp. 279.