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M M Palcic

Publications and source records attributed to M M Palcic.

At least 73 records · Page 4Linked to original sources
PubMed1995

Acceptor specificity of different length constructs of human recombinant alpha 1,3/4-fucosyltransferases. Replacement of the stem region and the transmembrane domain of fucosyltransferase V by protein A results in an enzyme with GDP-fucose hydrolyzing activity.

The acceptor specificity of recombinant full-length, membrane-bound fucosyltransferases, expressed in COS-7 cells, and soluble, protein-A chimeric forms of alpha 1,3-fucosyltransferase (Fuc-T) III, Fuc-TIV, and Fuc-TV was analyzed toward a broad panel of oligosaccharide, glycolipid, and glycoprotein substrates. Our results on the full-length enzymes confirm and extend previous studies. However, chimeric Fuc-Ts showed increased activity toward glycoproteins, whereas chimeric Fuc-TIII and Fuc-TV had a decreased activity with glycosphingolipids, compared to the full-length enzymes. Unexpectedly, chimeric Fuc-TV exhibited a GDP-fucose hydrolyzing activity. In substrates with multiple acceptor sites, the preferred site of fucosylation was identified. Fuc-TIII and Fuc-TV catalyzed fucose transfer exclusively to OH-3 of glucose in lacto-N-neotetraose and lacto-N-tetraose, respectively, as was demonstrated by 1H NMR spectroscopy. Thin layer chromatography immunostaining revealed that FucT-IV preferred the distal GlcNAc residue in nLc6Cer, whereas Fuc-TV preferred the proximal Gl-cNAc residue. Incubation of Fuc-TIV or Fuc-TV with VI3NeuAcnLc6Cer resulted in products with the sialyl-LewisX epitope as well as the VIM-2 structure. To identify polar groups on acceptors that function in enzyme binding, deoxygenated substrate analogs were tested as acceptors. All three Fuc-Ts had an absolute requirement for a hydroxyl at C-6 of galactose in addition to the accepting hydroxyl at C-3 or C-4 of GlcNAc.

Animals↗
PubMed1995

Fluorescence-based enzymatic assay by capillary electrophoresis laser-induced fluorescence detection for the determination of a few beta-galactosidase molecules.

beta-Galactosidase can be assayed by monitoring the generation of the fluorescent products, fluorescein-mono-beta-D-galactopyranoside and fluorescein, when the fluorogenic substrate fluorescein-di-beta-D-galactopyranoside is used. We have used capillary electrophoresis with ultrasensitive laser-induced fluorescence detection to monitor the formation of the fluorescent products of off-column enzymatic reactions. By analyzing as little as 40 pl of the enzymatic mixture we obtain limits of detection of 6.5 x 10(-14) M beta-galactosidase or 1.6 molecules based on the detection of fluorescein-mono-beta-D-galactopyranoside.

Electrophoresis↗
PubMed1995

Stereochemistry of benzylamine oxidation by copper amine oxidases.

Copper amine oxidases (EC 1.4.3.6) exhibit stereochemical heterogeneity in their reaction specificities. Enzymes isolated from different sources have previously been shown to catalyze the deamination of tyramine and dopamine with abstraction of the pro-R hydrogen at C-1, the pro-S hydrogen, and net nonstereo-specific protein abstraction. In this study we report on the stereochemical course of proton removal from benzylamine for six copper amine oxidases using stereo-specifically deuterated benzylamines prepared by a combined chemical-enzymatic method. The product benzaldehydes from the amine oxidase reactions were reduced in situ with alcohol dehydrogenase and NADH providing benzyl alcohols which were analyzed by 1H NMR spectroscopy. The amine oxidases isolated from pea seedlings and bovine, horse, porcine, rabbit, and sheep plasma all react with abstraction of the pro-S hydrogen of benzylamine, irrespective of the stereochemical course of the oxidation of tyramine or dopamine. We also report that the enzymes isolated from horse and rabbit plasma contain 2,4,5-trihydroxyphenylalanine (topaquinone) as an organic cofactor based on visible absorbance spectroscopy of p-nitrophenylhydrazine-derivatized enzymes.

Amine Oxidase (Copper-Containing)↗
PubMed1995

Synthesis and biological activity of oligosaccharide libraries.

Random glycosylation has proven remarkably effective for the generation of mixtures of oligosaccharides. Clearly, not all of the possible glycosidically-linked isomers are formed in equal quantity in these reactions. In the instances where product structures have been thoroughly investigated, however, all have been shown to be present. So far, only one random glycosylation step has been performed and the challenge will be to see if two tandem steps can generate a useful oligosaccharide library. Whether or not the present formulation of random glycosylation succeeds as a dominant strategy for the synthesis of oligosaccharide libraries, this important challenge is open to many approaches where creativity in both formulating the problem, as well as experimentally addressing it, warrants a major international effort.

Carbohydrate Sequence↗
PubMed1995

Stereochemistry and cofactor identity status of semicarbazide-sensitive amine oxidases.

The relationship between the soluble copper topaquinone amine oxidases, the membrane bound semicarbazide-sensitive amine oxidases and lysyl oxidase remains unclear. The stereochemical course of substrate oxidation has been determined for each enzyme type and these studies suggest that SSAO and lysyl oxidase are closely related mechanistically, and that they are distinct from the copper amine oxidases. Both lysyl oxidase and SSAO catalyze the oxidation of tyramine with removal of the pro-S hydrogen from C-1 of this substrate. The copper amine oxidase enzymes that react with abstraction of the pro-S hydrogen from C-1 of substrates do not exhibit a solvent exchange pathway. In contrast, this exchange occurs in lysyl oxidase and SSAO reactions. The organic cofactor in all three enzyme types is a quinone; however, the spectral features of phenylhydrazine and p-nitrophenylhydrazine-derivatized SSAO differ from those reported for all known topaquinone-containing enzymes. Cofactor identification is further complicated by the lack of the characteristic topa motif, Asn-Tyr-Asp/Glu, in lysyl oxidase and the absence of any sequence information for SSAO.

Amine Oxidase (Copper-Containing)↗
PubMed1995

The biosynthesis of Lewis X in Helicobacter pylori.

The biosynthesis of the Lewis X determinant (Gal beta 1-4 [Fuc alpha 1-3]GlcNAc beta-) in three strains of Helicobacter pylori has been investigated. Strains UA 861, UA 802 and UA 1182 contain alpha 1,3 fucosyltransferase and beta 1,4 galactosyltransferase activities that synthesize the Lewis X structure by the transfer of monosaccharides from GDP-fucose and UDP-galactose donors, respectively. The enzyme reaction products that formed were characterized by capillary zone electrophoresis and by 1H-NMR spectroscopy. The biosynthetic pathway is therefore identical to that found in humans. In the three strains, the fucosyltransferase and galactosyltransferase activities differed in various cellular fractions. Km values for their donor and acceptor substrates also differed.

Carbohydrate Sequence↗
PubMed1994

Synthesis of a di-O-methylated pentasaccharide for use in the assay of N-acetylglucosaminyltransferase III activity.

The biantennary oligosaccharide analogue beta-D-Glc pNAc-(1-->2)-alpha-D-Man p-(1-->3)-[beta-D-Glc pNAc-(1-->2)-alpha- D-Man p-(1-->6)]-beta-D-man p-O(CH2)8COOMe (3) is a potential substrate for N-acetylglucosaminyltransferases (GlcNAcTs) III-V which are present in mammalian cells. The di-O-methylated analogue of 3, beta-D-Glc pNAc-(1-->2)-[4-O-methyl-alpha-D-Man p]-(1-->3)-[beta-D-Glc pNAc-(1-->2)-[6-O-methyl-alpha-D-Man p]-(1-->6)]-beta-D-Man p-O-(CH2)8COOMe (5), was prepared by a block synthesis approach involving sequential addition of two O-methylated disaccharide donors to a protected central beta-D-Man residue. The OH groups acted on by GlcNAcT-IV and -V are protected from glycosylation in 5 since they are present as their methyl ethers. Pentasaccharide 5 was found to be an excellent substrate for GlcNAcT-III (EC 2.4.1.144) from rat kidney with Km = 0.15 mM. The product formed by incubation of 5 with a rat kidney extract, in the presence of UDP-GlcNAc, was isolated, structurally characterized by NMR spectroscopy and confirmed to be the expected di-O-methyl hexasaccharide where a beta-D-Glc pNAc residue had been added to OH-4 of the central beta-D-Man p unit.

Animals↗
PubMed1994

Biosynthesis of Rhizobium meliloti lipooligosaccharide Nod factors: NodA is required for an N-acyltransferase activity.

Rhizobium bacteria synthesize N-acylated beta-1,4-N-acetylglucosamine lipooligosaccharides, called Nod factors, which act as morphogenic signal molecules to legume roots during development of nitrogen-fixing nodules. The biosynthesis of Nod factors is genetically dependent upon the nodulation (nod) genes, including the common nod genes nodABC. We used the Rhizobium meliloti NodH sulfotransferase to prepare 35S-labeled oligosaccharides which served as metabolic tracers for Nod enzyme activities. This approach provides a general method for following chitooligosaccharide modifications. We found nodAB-dependent conversion of N-acetylchitotetraose (chitotetraose) monosulfate into hydrophobic compounds which by chromatographic and chemical tests were equivalent to acylated Nod factors. Sequential incubation of labeled intermediates with Escherichia coli containing either NodA or NodB showed that NodB was required before NodA during Nod factor biosynthesis. The acylation activity was sensitive to oligosaccharide chain length, with chitotetraose serving as a better substrate than chitobiose or chitotriose. We constructed a putative Nod factor intermediate, GlcN-beta 1,4-(GlcNAc)3, by enzymatic synthesis and labeled it by NodH-mediated sulfation to create a specific metabolic probe. Acylation of this oligosaccharide required only NodA. These results confirm previous reports that NodB is an N-deacetylase and suggest that NodA is an N-acyltransferase.

Acetylglucosamine↗
PubMed1994

A continuous spectrophotometric assay for glycosyltransferases.

This paper describes a continuous spectrophotometric assay for glycosyltransferases. In this assay, a nucleotide diphosphate is coupled to NADH oxidation via pyruvate kinase and lactate dehydrogenase. The nucleotide diphosphate is produced either directly during the glycosyltransferase mediated reaction, or indirectly by the production of a nucleotide monophosphate during the glycosyltransferase mediated reaction, and subsequent conversion of the nucleotide monophosphate to nucleotide diphosphate using nucleoside monophosphate kinase. Using this assay, kinetic parameters for fucosyl-, sialyl-, and N-acetylglucosaminyltransferases were determined. The assay not only allows continual monitoring of the enzymatic reaction, but is rapid and allows the processing of 96 samples at once since it is performed in 96-well microtiter plates. In addition, the procedure provides a means of monitoring the activity of these enzymes using sugar-nucleotide donor analogs, where radiochemical procedures cannot be used.

Cytidine Monophosphate N-Acetylneuraminic Acid↗
PubMed1994

A spectrophotometric assay for glucosidase I.

A spectrophotometric assay for glucosidase I using the synthetic trisaccharide alpha-D-Glc 1-->2 alpha-D-Glc 1-->3 alpha-D-Glc-O(CH2)8COOCH3 is reported. The terminal glucose is released from the substrate by the enzyme and quantitated using glucose oxidase, peroxidase, and o-dianisidine. The trisaccharide is specific for glucosidase I and provides all the necessary structural features for correct interaction in the enzyme active site. The utility of the assay for monitoring enzyme activity during isolation and for use in kinetic and inhibition studies (i.e., with 1-deoxynorjirimycin) is demonstrated.

1-Deoxynojirimycin↗
PubMed1994

Key involvement of all three GlcNAc hydroxyl groups in the recognition of beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->6)-beta-D-Glcp-OR by N-acetylglucosaminyltransferase-V.

N-Acetylglucosaminyltransferase-V (GlcNAc T-V) transfers a beta-linked GlcNAc residue from UDP-GlcNAc to OH-6' (of the alpha Man residue) in oligosaccharides terminating in the sequence beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->6)-beta-D-Glcp(or Manp)-OR (3, R = (CH2)7CH3). It was previously found that OH-4" (of the GlcNAc residue) in 3 was a critical element for substrate recognition by this enzyme. We show here that OH-3" and OH-6" are also key recognition elements. Four analogs of trisaccharide 3 where OH-3" and OH-6" were replaced, independently, by NH2 and NHAc groups, were prepared by multi-step chemical synthesis and kinetically evaluated as substrates for GlcNAc T-V from hamster kidney. These substitutions were selected since they replaced the OH groups with groups probing both hydrogen bonding and steric bulk. The 3"-modified compounds were found to be very poor substrates with Km values more than 50-fold elevated over that for 3 (26 microM) while the 6"-modified compounds were completely inactive. An intact 3,4,6 triol system in the terminal GlcNAc residue therefore appears to be the key polar group system that is recognized by this enzyme.

Acetylglucosamine↗
PubMed1994

Detection of 100 molecules of product formed in a fucosyltransferase reaction.

We present the detection of 100 molecules of enzyme substrate and product. A fucosyltransferase and a fucosidase were used to add and remove, respectively, a monosaccharide from a synthetic oligosaccharide fluorescently labelled with tetramethylrhodamine. The reaction was followed by use of capillary zone electrophoresis, to separate the product and reactant, with laser-induced fluorescence detection. These are the most sensitive enzyme assays reported to date and six orders of magnitude more sensitive than any reported for these two enzymes. This simple technology allows high-sensitivity determination of the activity of any enzyme for which a fluorescent substrate can be synthesized, and brings within reach the ability to assay glycosyltransferase activities in single cells.

Carbohydrate Conformation↗
PubMed1993

Substrate recognition by amyloglucosidase: evaluation of conformationally biased isomaltosides.

Amyloglucosidase catalyzes the hydrolysis of methyl beta-maltoside (1) 30-50 times more rapidly than methyl alpha-isomaltoside (2). It is established that OH-6', OH-4', and OH-4 which are involved in key polar interactions with the enzyme in the case of isomaltoside. Conformational analyses based on HSEA calculations indicate that the dispositions in space of OH-3 of maltose relative to OH-4' and OH-6' in the preferred conformation for the maltoside (1) is energetically more readily achieved by methyl 6R-C-methyl-alpha-isomaltoside (3), than for its 6-S-isomer (4). A kinetic evaluation of the hydrolysis in fact has shown that the R-compound is more strongly bound by the enzyme (Km = 0.9 mM) than the parent isomaltoside (Km = 24.5 mM), whereas the S-compound has the weakest enzyme binding (Km = 90 mM). Since the kcat values were all within the range 0.85 +/- 0.20 s-1, it is evident that the relative rates of hydrolysis are related to the relative ease for the compounds to achieve an interaction of a hydroxyl group in the aglycon of an alpha-D-glucopyranoside with the enzyme for the formation of the enzyme-substrate complex. The relative rates of hydrolysis of the alpha-glucosides of the 1,3-dihydroxy-trans-decalins, 5 and 6, provide further support for this highly desirable but not necessary recognition for the orientation of the reducing glucose unit in the active site.

Carbohydrate Conformation↗
PubMed1993

Chemical synthesis and kinetic characterization of UDP-2-deoxy-D-lyxo-hexose("UDP-2-deoxy-D-galactose"), a donor-substrate for beta-(1-->4)-D-galactosyltransferase.

Bovine beta-(1-->4)-galactosyltransferase (GalT) transfers galactose from UDP-galactose to beta-D-GlcpNAc-terminating oligosaccharides to produce N-acetyllactosamine sequences. We report here the chemical synthesis, structural characterization and enzymatic evaluation of the very labile UDP-2-deoxy-D-lyxo-hexose ("UDP-2-deoxy-galactose," 2) as an alternate donor for GalT. Donor 2 had kinetic parameters, including a Km value of 51 microM, almost identical to those for the natural substrate UDP-galactose when beta-D-GlcpNAc-O(CH2)8COOMe was used as the acceptor. The product of the enzymatic transfer was isolated and confirmed to have the expected 2'-deoxy-N-acetyllactosamine sequence.

Animals↗