N-acetylneuraminic acid and sialoglycoconjugate metabolism in fibroblasts from a patient with generalized N-acetylneuraminic acid storage disease

Cultured skin fibroblasts from a patient suffering from generalized N-acetylneuraminic acid storage disease were found to accumulate large amounts (approx. 4.0 μmol/g fresh weight) of free N-acetylneuraminic acid in a lysosome-enriched subcellular fraction. However, there were no detectable deficiencies in lysosomal hydrolase activities (including neuraminidase), and the activities of CMP-N-acetylneuraminic acid synthetase and N-acetylneuraminic acid aldolase were within normal limits. The cellular glycoconjugate composition was normal, and pathologic fibroblasts labeled with either [³H]glucosamine-HCl or $\text{N-[}{}^3\text{H]acetylmannosamine}$ showed a marked accumulation of labeled free N-acetylneuraminic acid, along with elevated incorporation into sialoglycoconjugates. Neither normal nor pathologic fibroblasts secreted labeled free N-acetylneuraminic acid into the culture medium. These results are consistent with an inherited defect in N-acetylneuraminic acid reutilization, resulting in the lysosomal accumulation of the free monosaccharide in generalized N-acetylneuraminic acid storage disease.     

Conversion of N-acetylglucosamine to CMP-N-acetylneuraminic acid in a cell-free system of rat liver

A soluble fraction of rat liver converts glucosamine and N-acetylglucosamine in the presence of ATP and UTP to N-acetylneuraminic acid. This system, when supplemented with CTP, forms CMP-N-acetylneuraminic acid in high yield. Nicotinamide was found to enhance the synthesis of UDP-N-acetylglucosamine and N-acetylneuraminic acid. Kinetic analysis reveals N-acetylglucosamine 6-phosphate, UDP-N-acetylglucosamine, N-acetylmannosamine, N-acetylmannosamine 6-phosphate and N-acetylneuraminic acid 9-phosphate as intermediates. Under certain experimental conditions, however, an epimerisation of N-acetylglucosamine to N-acetylmannosamine was seen.     

Affinity chromatography of nucleosides and nucleic acid base derivatives with nucleic acid bases or nitrobenzeneboronic acid substituted silicas

Nucleic acid bases such as adenine and uracil, and nitrobenzeneboronic acid substituted silicas were prepared by the reaction of chloromethylbenzene substituted silica with adenine sodium salt and trimethylsilylated uracil, and nitration of benzeneboronic acid substituted silica, respectively. From the results of HPLC of nucleosides and N-ethyl derivatives of nucleic acid bases using modified silicas, hydrophobic base stacking interaction, selective hydrogen bonding interaction between purine and pyrimidine bases, and reversible cyclic boronate ester formation between diols of nucleosides with boronic acid were effective for the separation of nucleic acid related compounds. Moreover, association constants for hydrogen bonding formation of nucleic acid bases were estimated.     

The synthesis and antistaphylococcal activity of N-sulfonaminoethyloxime derivatives of dehydroabietic acid

A series of N-sulfonaminoethyloxime derivatives of dehydroabietic acid were synthesized and investigated for their antibacterial activity against Staphylococcus aureus Newman strain and multidrug-resistant strains (NRS-1, NRS-70, NRS-100, NRS-108 and NRS-271). Most of the target compounds having chloro, bromo, trifluoromethyl phenyl moiety exhibited potent in vitro antistaphylococcal activity. The meta-CF₃ phenyl derivative T23 showed the highest activity with MIC of 0.39–0.78?μg/mL against S. aureus Newman, while several analogues showed similar potent antibacterial activity with MIC values between 0.78 and 1.56?μg/mL against five multidrug-resistant S. aureus. The stability of T35 in plasma of SD rat and the cellular cytotoxicity were also evaluated.     

5′-Uridyl derivatives of N-glycosyl allophanic acid and biuret

(2′,3′-O-Isopropylidene-5′-uridyl) 4-(2,3,4,6-tetra-O-acetyl-β-d-glycopyranosyl)allophanates were obtained in the reactions of 2′,3′-O-isopropylidene-uridine and O-peracetylated β-d-gluco-, galacto- and xylopyranosylamines, and OCNCOCl. 2,3,4,6-Tetra-O-acetyl-β-d-glucopyranosyl isocyanate and N-(2′,3′-O-isopropylidene-5′-uridyl)urea gave 1-(2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl)-5-(2′,3′-O-isopropylidene-5′-uridyl)biuret. Deprotection of the β-d-gluco configured allophanate and biuret was carried out by standard methods.     

Properties and regional distribution of cerebral CMP-N-acetylneuraminic acid: Glycoprotein sialyltransferase

Glycoprotein sialyltransferase was studied in the rat brain and in the frontal grey cortex and corpus callosum of the calf brain. Activities were measured with endogenous acceptors as well as with desialized α₁-acid glycoprotein as an exogenous acceptor. The enzyme was characterized by means of its pH optimum, K_m values and requirements for detergent and cations. The properties of the rat and calf brain enzymes appeared to be very similar. Substrate specificity studies indicate that more than one glycoprotein sialyltransferase reaction may occur in brain. The regional distribution of the enzyme in the calf brain was rather uniform. From this it was concluded that glycoprotein sialyltransferase, at least for the greater part, is localized in membranes other than those of the synaptic complexes, and occurs in both neurons and glia cells. The regional distribution of the amounts of endogenous glycoprotein acceptor sites, which could be calculated from the sialyltransferase activities, showed a striking correlation with that of the protein-bound sialic acid, but not with the sialyltransferase activity. The role of these endogenous glycoprotein acceptors in cerebral sialoglycoprotein biosynthesis is discussed.     

Affinity chromatography of thiol-containing purines and ribonucleic acid.

Evidence is presented for the specific interaction of 6-mercaptopurine with mercurated cellulose. Following from this, a new method is described for the affinity chromatography of thiol-containing molecules and of RNA containing incorporated 6-thioguanosine on columns of mercurated cellulose. This technique may find application in the study of RNA metabolism and gene expression.     

N,N-Dialkylcarbamato derivatives of niobium and tantalum as precursors to metal-functionalized silica surfaces

Chemical implantation of Group 5 cations [Nb(III), Nb(V), and Ta(V)] has been carried out under mild conditions by the reaction of N,N-dialkylcarbamato derivatives M(O₂CNR₂)_n (M = Nb, Ta) with silanol groups of amorphous silica, carbon dioxide, and secondary amine being released in the process. The amount of supported cations depends on the metal and on the initial number of N,N-dialkylcarbamato ligands on M; partial reduction to the +4 oxidation state occurs in the case of Nb(O₂CNR₂)₅.     

Dianthramides A and B,two N-benzoylanthranilic acid derivatives from elicited tissues of Dianthus caryophyllus

Two new phenolic derivatives, dianthramide A and B, were isolated from Dianthus caryophyllus tissues elicited with mycelial extracts of Phytophthora parasitica. The purified substances were identified on the basis of their spectral data and were characterized as N-salicyl-4-methoxyanthranilic acid (dianthramide A) and N-salicyl-4-hydroxyanthranilic acid methyl ester (dianthramide B). Dianthramides A and B co-occur in carnation tissues with the known phytoalexin dianthalexin.     

Affinity chromatography on immobilised triazine dyes. Studies on the interaction with multinucleotide-dependent enzymes

A systematic investigation into the interaction of several triazinyl dyes with two enzymes from purine metabolism, IMP dehydrogenase (IMP: NAD+ oxidoreductase, EC 1.2.1.14( and adenylosuccinate synthetase (IMP: L-aspartate ligase (GDP-forming), EC 6.3.4.4) has been conducted. Evidence from kinetic inhibition studies, enzyme inactivation with specific affinity labels and specific elution techniques from agarose-immobilised dyes indicate that triazine dyes such as Procion Blue H-B (Cibacron Blue F3G-A), Red HE-3B and Red H-3B are able to differentiate between the nucleotide-binding sites of these enzymes. This information has been exploited to design specific elution techniques for the purification of these enzymes by affinity chromatography.     

Affinity chromatography of Aspergillus acid proteinase on an N,O-dibenzyloxycarbonyl-tyrosine-AH-Sepharose 4B column

N,O-dibenzyloxycarbonyl-l-tyrosine Z-Tyr(Z) has been found to be a specific inhibitor of Aspergillus acid proteinase and was coupled with hexamethylene-diamino-Sepharose 4B (AH-Sepharose 4B). By affinity chromatography employing a Z-Tyr(Z)-AH-Sepharose 4B column, acid car?ypeptidase-free Aspergillus acid proteinase was obtained.     

Affinity purification of seminalplasmin and characterization of its interaction with calmodulin.

Bull seminalplasmin antagonizes with high potency and selectivity the activating effect of calmodulin on target enzymes [Gietzen & Galla (1985) Biochem. J. 230, 277-280]. In the present paper we establish that seminalplasmin forms a 1:1, Ca2+-dependent and urea-resistant complex with calmodulin. The dissociation constant equals 1.6 nM. In the absence of Ca2+ a low-affinity complex is formed that is disrupted by 4 M-urea. On the basis of these properties, a fast affinity purification of seminalplasmin was developed. The high specificity of seminalplasmin as a calmodulin antagonist was demonstrated for the multipathway-regulated adenylate cyclase of bovine cerebellum. Far-u.v. c.d. properties are consistent with a random form of seminalplasmin in aqueous solution; 23% alpha-helix is induced on interaction with calmodulin. The fluorescence properties of the single tryptophan residue of seminalplasmin are markedly changed on formation of the complex. These studies allowed us to locate tentatively the peptide segment that interacts with calmodulin, and to ascertain the structural homology between seminalplasmin and other calmodulin-binding peptides. Additional material, showing the inhibition of calmodulin-mediated activation of bovine brain phosphodiesterase by melittin and seminalplasmin and also the near-u.v. spectrum of affinity-purified seminalplasmin, has been deposited as supplement SUP 50135 (4 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1986) 233, 5.     

Chemical implantation of Group 4 cations on silica via cyclopentadienyl- and N,N-dialkylcarbamato derivatives

Chemical implantation of Group 4 cations [Ti(III), Ti(IV), Zr(IV), Hf(IV)] has been carried out under mild conditions by the reaction of polycyclopentadienyl- (MCp_n; M = Ti, n = 3, 4; M = Zr, Hf, n = 4), mixed cyclopentadienyl/N,N-dialkylcarbamato (ML_x(O₂CNEt₂)_y; M = Ti, L = Cp, C₅Me₅ (Cp*), x = 2, y = 1; M = Hf, L = Cp, x = 1, y = 3), and N,N-dialkylcarbamato (M(O₂CNR₂)_n, M = Ti, n = 3, R = ⁱPr; M = Ti, Hf, n = 4, R = Et; M = Zr, n = 4, R = ⁱPr) derivatives, with the silanol groups of amorphous silica. Cyclopentadiene/pentamethylcyclopentadiene and/or carbon dioxide and the secondary amine are released in the process. The amount of implanted cations depends on the metal and on the ligands, the pentamethylcyclopentadienyl complex being less reactive than the unsubstituted congener. The starting complexes and the final products have been characterized by EPR or by ¹³C CP-MAS NMR spectroscopy.     

Determination of serotonin,its precursors,metabolites and [3H]serotonin in lung by high-performance liquid chromatography with fluorescence detection

A rapid, simple, sensitive method for the determination of serotonin, its precursors, metabolites and [³H]serotonin in lung is described. Tissue preparation requires only homogenization in dilute perchloric acid and centrifugation prior to separation by high-performance liquid chromatography using a reversed-phase column. Detection is based on the native fluorescence of indole compounds and detection limits ranged between 30–90 pg injected. The method has been used to determine these compounds in mouse lung and plasma.     

Affinity labeling of the rabbit 12/15-lipoxygenase using azido derivatives of arachidonic acid

Lipoxygenases are lipid-peroxidizing enzymes, which have been implicated in the pathogenesis of important diseases. They consist of a single polypeptide chain, which is folded into a two-domain structure. The large catalytic domain contains the putative substrate-binding pocket and the catalytic non-heme iron. To identify structural elements of the rabbit 12/15-lipoxygenase that are involved in enzyme/substrate and/or enzyme/product interaction, we synthesized a set of radioactively labeled lipoxygenase substrates carrying a photoreactive azido group (17-azido-ETE, 18-azido-ETE, 19-azido-ETE) and used these compounds as affinity probes. After photoaffinity labeling, the enzyme was digested proteolytically and modified tryptic cleavage peptides were identified by a combination of radio-HPLC and mass spectral analysis. Following this strategy, we observed covalent linkage of a cleavage peptide that contained Ile593, which has previously been identified as the sequence determinant for the positional specificity. These data are consistent with the previous suggestion that this peptide lines the substrate-binding pocket. Surprisingly, we also observed strong labeling of cleavage peptides originating from the N-terminal beta-barrel domain, and our mass spectral data suggested covalent linkage of oxidized affinity probes. Taken together, these results confirm the previous conclusion that Ile593 and surrounding amino acids are constituents of the active site, but they also implicate the N-terminal beta-barrel in enzyme/substrate and/or enzyme/product interaction.