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.

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.

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.

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 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Separation and some properties of D-galactonate pathway enzymes from Mycobacterium sp. 607.

D-galactonate-grown cells of Mycobacterium sp. 607 can utilize D-galactonate by a pathway involving D-galactonate dehydratase, 2-keto-3-deoxy-galactonate kinase and 6-phospho-2-keto-3-deoxygalactonate aldolase. The enzymes have been separated by ion-exchange chromatography on DEAE-cellulose or ultrafiltration on Sephadex G-100.

A galactokinase of Mycobacterium sp. 279 galactose mutant.

A galactokinase and the other enzymes of a galactose catabolic pathway were found in Mycobacterium sp. 279 galactose mutant. The galactokinase was partially purified in a procedure involving ammonium sulfate precipitation, Sephadex G-100 filtration and DEAE-cellulose chromatography.