Determination of neuraminidase-susceptible and total N-acetylneuraminic acid in cells by selected ion monitoring.

N-acetylneuraminic acid (NANA) is released from glycoproteins by neuraminidase or acid hydrolysis and quantified by monitoring the protonated molecular ions of fully silylated NANA ($\text{m}\text{e}\text{= 814}$) and neuraminic acid β-methylglycoside (internal standard, $\text{m}\text{e}\text{= 714}$) in a gas chromatograph—mass spectrometer system using isobutane ionization. Detection limit is 200 pg (0.6 pmol, underivatized weight) of NANA injected. In biological samples 5 ng (15 pmol) of NANA can be detected in 50 μl of hydrolysate. Only 1 to 50 μl of hydrolysate is needed, sample preparation is simple, NANA is positively identified in every analysis, 2-deoxy carbohydrates and other sialic acids do not interfere, only free NANA is determined, and the internal standard increases reliability. The NANA content of neuraminidase-treated human leukemic cells was on the order of $\text{0.3μ}\text{mol}\text{10}{}^9$ cells. NANA was quantified using as few as 5 × 10⁴ cells, in contrast to the conventional colorimetric (thiobarbituric) technique which requires 2.5 × 10⁷ cells.     

Peroxidase-amplified assay of sialidase activity toward gangliosides.

Sialidase assays were carried out with the substrate, ganglioside GD1a, coated onto enzyme immunoassay plate wells. Following the incubation of GD1a with sialidase from V. cholerae, the amount of ganglioside GM1 produced was measured as follows: cholera toxin B subunit conjugated to horseradish peroxidase was added to specifically bind to GM1, and then the amount of bound peroxidase was determined in a colorimetric enzymatic assay. In the absence of detergent, linearity for the detection of GM1 was 0 to 0.5 pmol per well, and the sensitivity for sialidase detection was about 3 fmol of product formed per minute. The addition of detergent (Triton CF-54) to the assay reduced the sensitivity and increased the amount of substrate required. Application of this assay for the detection of cell-derived neutral (pH 6.5) sialidase activities in the conditioned medium of human skin fibroblasts is described.     

Phase-transfer-catalysed synthesis of N-acetylneuraminic acid alpha-thioketosides and inhibitor studies with Clostridium perfringens sialidase

The alpha-thioketosides of methyl 5-acetamido-4,7,8,9-tetra-O-acetylneuraminate with thioacetic acid, thiophenol, 4-nitrothiophenol, 4-aminothiophenol, 2-mercaptopyridin and mercaptobenzothiazol as aglycones were synthesized by phase-transfer catalysis in good yields. The methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-2-thioacetylneuraminate is the analogous thio compound to the methyl 5-acetamido-2,4,7,8,9-penta-O-acetylneuraminate and can be used as intermediate for preparing S-ketosides of Neu5Ac. By Zemplen saponification and mild hydrolysis of the methyl-ester group the free Neu5Ac-alpha-thioketosides with thiophenol, 4-nitrothiophenol, 4-aminothiophenol and 2-mercaptopyridin could be prepared. These ketosides were found to be inhibitors of C. perfringens sialidase with Ki-values between 2.3mM and 6.6mM. The free Neu5Ac-alpha-mercaptobenzothiazolyl ketoside could not be prepared by this procedure. It was completely hydrolysed during Zemplen saponification and methyl-ester hydrolysis in alkaline medium.     

Characterization of cytosolic sialidase from Chinese hamster ovary cells: part II. Substrate specificity for gangliosides

Cytosolic Chinese hamster ovary (CHO) cell sialidase has been cloned as a soluble glutathione S-transferase (GST)-sialidase fusion protein with an apparent molecular weight of 69 kD in Escherichia coli. The enzyme has then been produced in mg quantities at 25-L bioreactor scale and purified by one-step affinity chromatography on glutathione sepharose (Burg, M.; Müthing, J. Carbohydr. Res. 2001, 330, 335-346). The cloned sialidase was probed for desialylation of a wide spectrum of different types of gangliosides using a thin-layer chromatography (TLC) overlay kinetic assay. Different gangliosides were separated on silica gel precoated TLC plates, incubated with increasing concentrations of sialidase (50 degreesU/mL up to 1.6 mU/mL) without detergents, and desialylated gangliosides were detected with specific anti-asialoganglioside antibodies. The enzyme exhibited almost identical hydrolysis activity in degradation of GM3(Neu5Ac) and GM3(Neu5Gc). A slightly enhanced activity, compared with reference Vibrio cholerae sialidase, was detected towards terminally alpha(2-3)-sialylated neolacto-series gangliosides IV3-alpha-Neu5Ac-nLc4Cer and VI3-alpha-Neu5Ac-nLc6Cer. The ganglio-series gangliosides G(D1a), G(D1b), and G(T1b), the preferential substrates of V. cholerae sialidase for generating cleavage-resistant G(M1), were less suitable targets for the CHO cell sialidase. The increasing evidence on colocalization of gangliosides and sialidase in the cytosol strongly suggests the involvement of the cytosolic sialidase in ganglioside metabolism on intracellular level by yet unknown mechanisms.     

Biosynthesis of gangliosides in primary cultures of rat hepatocytes. Determination of the net synthesis of individual gangliosides by incorporation of labeled N-acetylmannosamine

The ganglioside content of rat hepatocytes increases several-fold during the first 6 days in monolayer culture. To correlate increased levels with rates of de novo synthesis, the incorporation of N-acetyl-[6-3H]D-mannosamine into individual gangliosides was determined. The calculation of synthetic rates was made possible by the simultaneous measurement of the specific radioactivity of the immediate sialic-acid donor, CMP-Neu5Ac. The CMP-Neu5Ac content of hepatocytes was found by HPLC analysis to be 30.5 nmol/g of plated cells. The specific radioactivity of this precursor pool reached a constant plateau 5 h after addition of the labeled N-acetyl-mannosamine and remained constant for at least 70 h. The incorporation into individual gangliosides was measured in primary cultures of rat hepatocytes between 72 and 144 h after seeding. During this period, the increase in ganglioside levels was greatest. The highest rates of incorporation were seen in GD1a followed by GM3, GM1, GD3 and the polysialylated compounds. The following rates of synthesis (nmol per 60 h and mg of protein) were calculated: GD1a 0.68, GM3 0.59, GM1 0.36, GD3 0.13 and GT1 0.02. These values are compared with the net increase of the gangliosides as measured by the resorcinol reaction.     

Degradation of gangliosides by the lysosomal sialidase requires an activator protein.

Lysosomal sialidase, which was formerly believed to degrade only water-soluble substrates but not glycolipids, cleaves ganglioside substrates II3NeuNAc-LacCer, IV3NeuNAc, II3NeuNAc-GgOse4Cer, IV3 NeuNAc, II3(NeuNAc)2-GgOse4Cer when these are dispersed either with an appropriate detergent (taurodeoxycholate) or with the sulfatide activator protein, a physiologic lipid solubilizer required for the lysosomal hydrolysis of other glycolipids by water-soluble hydrolases. In the presence of the activator protein, time and protein dependence were linear within wide limits, while the detergent rapidly inactivated the enzyme. The disialo group of the b-series gangliosides was only poorly attacked by the enzyme when the lipids were dispersed with the activator protein, whereas in the presence of the detergent, they were hydrolyzed as fast as terminal sialic acid residues. With the appropriate assay method, significant ganglioside sialidase activity could be demonstrated in the secondary lysosome fraction of normal skin fibroblasts but not of sialidosis fibroblasts. Our results support the notion that there is only one lysosomal sialidase, which degrades both the water-soluble and the membrane-bound sialyl glycoconjugates.     

Multiple deoxyribonuclease activities in nuclei of HeLa cells

Extracts of purified HeLa nuclei contain DNase activities which can be separated by CM-Sephadex column chromatography into four distinct enzymes. These DNases differ in their resistance to thermal inactivation and relative activity on different substrates. Each of these DNases can increase the template-primer activity of native DNA for DNA polymerase action.     

Mobilization of iron from specifically labeled reticulocyte ghosts.

Specifically labeled 59Fe ghosts have been prepared by incubation of whole reticulocytes with 59Fe3+-transferrin-CO3(2)-- followed by washing and ghost isolation. The binding of 59Fe by the membrane fraction is quite stable over a wide range of conditions, but iron mobilization occurs on incubation with chelating agents or cell lysate. The time course of 59Fe mobilization by unlabeled reticulocyte lysate exhibits five apparently zero-order phases. The rate of iron mobilization is linearly dependent on the concentration of 59Fe ghosts present in the incubation mixture. In contrast, the relative concentration of lysate appears to exhibit a saturation dependence with regard to membrane iron mobilization. Bathophenanthroline sulfonate follows a multiphasic time course of iron mobilization similar to that found with the lysate. Lysate from mature erythrocytes was found to mobilize iron with kinetics that are identical to reticulocyte lysate. The number and duration of the phases is independent of the mobilizing agent. The role of the membrane fraction in regulating the rate of iron release to cytosol was also investigated by the repetitive incubation of 59Fe ghosts with fresh lysate. The rate of 59Fe mobilization depended on the condition of the ghost with regard to prior 59Fe depletion. This publication emphasizes the active role of the membrane fraction in determining the rate at which iron will become available to the cytosol and the possibility that cytosol factors modulate the action of membrane bound components.     

Interactions of pig brain cytosolic sialidase with gangliosides. Formation of catalytically inactive enzyme-ganglioside complexes

Cytosolic sialidase A was extracted from pig brain and purified about 2000-fold with respect to the starting homogenate (about 550-fold relative to the cytosolic fraction). The enzyme preparation provided a single peak on Ultrogel AcA-34 column chromatography and had an apparent molecular weight of 4 x 10(4). On incubation with micellar ganglioside GT1b, (molecular weight of the micelle, 3.5 x 10(5)) under the conditions used for the enzyme assay, brain cytosolic sialidase A formed two ganglioside-enzyme complexes, I and II, which were isolated and characterized. Complex II had a molecular weight of 4.2 X 10(5), and a ganglioside/protein ratio (w/w) of 4:1. This is consistent with a stoichiometric combination of one ganglioside micelle and two enzyme molecules. Complex I was probably a dimer of complex II. In both complexes I and II cytosolic sialidase was completely inactive. Inactivation of cytosolic sialidase by formation of the corresponding complexes was also obtained with gangliosides GD1a and GD1b, which, like GT1b, are potential substrates for the enzyme and GM1, which is resistant to the enzyme action. Therefore, the enzyme becomes inactive after interacting with ganglioside micelles. GT1b-sialidase complexes acted as excellent substrates for free cytosolic sialidase, as did the complexes with GD1a and GD1b.     

RNA annealing activities in HeLa nuclei.

RNA-RNA base pairing plays a critical role in the interactions between pre-mRNAs and trans-acting factors during the processing of pre-mRNAs (hnRNAs) into mRNAs, and it is likely that specific factors are required to promote the annealing of RNAs. To identify particular nuclear components that have such activity, we fractionated HeLa nucleoplasm and assayed for activity which promoted the hybridization of a pre-mRNA with an antisense RNA probe complementary to 60 nucleotides (nt) encompassing the 3' splice site. At least nine major RNA annealing activities were identified and, surprisingly, eight of these copurified partially or to homogeneity with known hnRNP proteins. The activities of three of these proteins, hnRNP A1, C1 and U, were confirmed using purified recombinant proteins. Moreover, we found that the RNA binding domain alone of hnRNP C1/C2 had significant activity, indicating that this RNA annealing may result, at least partly, from chaperone activity: a direct modulation of RNA conformation by hnRNP proteins. The finding that hnRNP proteins have strong RNA annealing activity indicates that they can profoundly affect the interactions of pre-mRNAs with trans-acting factors and suggests this to be an important function of hnRNP proteins in the processing of pre-mRNAs.     

Determination of brain gangliosides by determination of ganglioside stearic acid

A new method is described for the determination of brain gangliosides by measuring stearic acid, the chief acid of gangliosides, in an appropriately purified brain extract. The method includes extraction of tissue with chloroform-methanol, extraction of gangliosides from the extract with 0.1 M KCl, evaporation of the aqueous phase, methanolysis, and gas-liquid chromatography of the resultant methyl esters with a double internal standard. The method depends on the simple composition of ganglioside fatty acids (80% stearic acid) and allows determination of less than 0.05 micromole of gangliosides. Interfering lipids are removed from the ganglioside extract by washing with chloroform-methanol-water. The effects of contamination with nonlipid N-acetylneuraminic acid are avoided.     

Rapid chemotaxis assay using radioactively labeled bacterial cells.

A rapid chemotaxis assay is described in which radioactively labeled cells of the assay organism are used to detect the number of cells trapped in capillaries containing attractant. The sensitivity and reproducibility of the radioactive technique is comparable to that of the dilution plating procedure of Adler (J. Adler, J. Gen. Microbiol. 17:77-91, 1973), but is faster and also permits the results of the assay to be determined on the day that the assay is run. The method could be particularly useful for environmental studies and for field experiments, since it does not rely on sterile techniques for dilution plating.     

The biosynthesis of gangliosides. The incorporation of galactose, N-acetylgalactosamine and N-acetylneuraminic acid into endogenous acceptors of subcellular particles from rat brain in vitro

Gangliosides bound to subcellular particles from rat brain were labelled by incubation of the particles (i) with CMP-N[(3)H]-acetylneuraminic acid and (ii) simultaneously, with CMP-N[(3)H]-acetylneuraminic acid and UDP-N-acetyl-[(14)C(1)]galactosamine or with CMP-N[(3)H]-acetylneuraminic acid and UDP-[U-(14)C]-galactose. Analysis of the labelled gangliosides showed that in (i), (a) the labelling was mostly in the neuraminidase-labile sialyl groups, (b) rigid relationships exist between the enzymes and the sialyl acceptors; the enzymes are not free to interact with all the specific substrates present in the preparation and (c) the precursor of the trisialoganglioside was the major disialoganglioside with a sialyl 2-->8 sialyl group. In (ii), (a) precursor-product relationships between the main pools of each ganglioside apparently do not exist, (b) for the labelling of Tay-Sachs ganglioside the amount formed from hematoside was at least 2.5 times that from aminoglycolipid and (c) the major monosialoganglioside was the precursor for the major disialoganglioside with a sialyl 2-->8 sialyl group.     

Heterogeneity of nuclear proteins from HeLa cells specifically binding to the Alu sequence

Nuclear proteins from HeLa cells specifically binding to the Alu-repeat cloned in the plasmid Blur8 have been studied. 0.35 M nuclear extract proteins have been separated on DEAE-cellulose. The presence of DNA-binding proteins has been found in all fractions by the technique of DNA-binding on nitrocellulose filters. The labelled restricted DNA of the plasmid Blur8 was incubated with the proteins of different fractions with the subsequent identification of specific Alu-protein complexes in polyacrylamide gel at low ionic strength. At least two proteins have been found to have the different affinity to Alu-repeat. Various functions of Alu-repeats and the possibility of their participation in the initiation of DNA replication are discussed.