Peroxidase-amplified assay of sialidase activity toward gangliosides

Ogura, Kiyoshi; Ogura, Misa; Anderson, Richard L.; Sweeley, Charles C.

doi:10.1016/0003-2697(92)90276-d

Cited by 17 publications

(5 citation statements)

References 18 publications

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“…The number of cells was calculated from the protein content of each aliquot of the homogenate by a modified Lowry method (Peterson, 1977). To determine GM1 content in the hepatocytes and the conditioned medium, enzymelinked immunosorbent assay ELISA on polystyrene microtest plates (Biomedical, Russia) were used; cholera toxin B-subunit peroxidase conjugate was used (Ogura et al, 1992;Ravindranath et al, 1994;Zvezdina et al, 2000). GM1 0.015-1.0 pmol in ethanol solution was used as an internal standard and a calibration curve drawn.…”

Section: Ganglioside Gm1 Contentmentioning

confidence: 99%

Calcium ions as a factor of cell—cell cooperation in hepatocyte cultures

Brodsky

Zvezdina

Nechaeva

et al. 2003

Cell Biology International

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Ultradian protein synthesis rhythm was used as a marker of cell cooperation in synchronous dense and non-synchronous sparse hepatocyte cultures. Phenylephrine (2 microM, 2 min), an alpha (1)-adrenoreceptor agonist, which exerts [Ca(2+)](cyt)elevation from intracellular stores, affected protein synthesis rhythm in sparse cultures, i.e. initiated cooperative activity of the cells. The same effect was produced by 2,5-di(tertiary-butyl)-1,4-benzohydroquinone (10 microM, 2 min), which increases [Ca(2+)](cyt)by a non-receptor pathway. Pretreatment of dense cultures with the intracellular calcium chelator, 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'- tetraacetic acid (acetoxymethyl) ester (BAPTA-AM) at 10-20 microM for, 30-60 min resulted in loss of the rhythm of protein synthesis, i.e. loss of cooperative activity between the cells. The medium conditioned by control dense cultures initiated rhythm in sparse cultures, whereas the conditioned medium of cultures pretreated with BAPTA-AM did not. [Ca(2+)](cyt)increase is known to occur with monosialoganglioside GM1 treatment. By ELISA estimation, the GM1 content in 3 h conditioned medium was similar in control dense cultures to that in cultures pretreated with BAPTA-AM. Bearing in mind data on the Ca(2+)-dependence of vesicle formation and shedding, the conditioned medium was separated by 150000 g centrifugation to supernatant containing monomers and micelles, and a pellet containing vesicular form of gangliosides. Only the latter initiated cooperative activity of the cells of sparse cultures. These cultures were also synchronized by GM1-containing liposomes at lower concentrations than added free GM1, 0.0003 and 0.06 microM respectively. Thus, GM1 and calcium are both involved in cell-cell synchronization. Activation of gangliosides, including GM1 and elevation of [Ca(2+)](cyt,)is known to lead to changes of protein kinase activity and protein phosphorylation resulting in modulation of oscillations in protein metabolism.

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Section: Ganglioside Gm1 Contentmentioning

confidence: 99%

Calcium ions as a factor of cell—cell cooperation in hepatocyte cultures

Brodsky

Zvezdina

Nechaeva

et al. 2003

Cell Biology International

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“…Polyacrylate, used as a highmolecular-weight carrier instead of BSA, is stable to chemical and proteolytic action. Carbohydrate-synthesizing and degrading enzymes often require detergents for their activity, which has been problematic in solid-phase assays employing glycolipids as substrates (23,24), because glycolipids are detached from plastic in the presence of detergents. The use of streptavidin as a universal capture reagent facilitates easy, rapid, and stable immobilization and makes it possible to choose the order of enzymatic reactions and immobilization.…”

Section: Discussionmentioning

confidence: 99%

“…Other disadvantages of the commonly used radiochemical methods (19,20) are production of radioactive waste and poor commercial availability of pure and highly active radioactive substrates. Previously described methods for measuring sialidase activity also include the use of fluorogenic substrates (21,22) and peroxidase-amplified solid-phase assays using glycolipids as substrates (23,24). The linkage specificity of sialidase is not revealed by the former methods.…”

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confidence: 99%

A Time-Resolved Immunofluorometric Assay of Sialyl Lewis x-Degrading α2,3-Sialidase Activity

Räbinä

Pikkarainen

Miyasaka

et al. 1998

Analytical Biochemistry

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“…76) In support of our hypothesis about the turnover of GM 3 in these cells, we found that GM 3 labeled with 14 C in the sialic acid residue and 3 H in the long-chain base (ceramide) lost the label preferentially in the sialic acid residue with little or no loss of label in the ceramide moiety. 76,77) …”

Section: Enzymes Of Glycosphingolipid Metabolismmentioning

confidence: 99%

Reflections on my career in analytical chemistry and biochemistry

Sweeley

2010

Proceedings of the Japan Academy. Ser. B: Physical and Biologic

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My career has been focused in two major areas, analytical chemistry and biochemistry of complex lipids and glycoconjugates. Included here are the pioneering work on the gas chromatography of long-chain sphingolipid bases, carbohydrates, steroids and urinary organic acids. Mass spectrometry was utilized extensively in structural studies of sphingolipids, fatty acids, carbohydrates, steroids, urinary organic acids, polyisoprenoid alcohols, and juvenile hormone. Computer systems were developed for the acquisition and analysis of mass spectra, and were used for development of automated metabolic profiling of complex mixtures of metabolites. Fabry’s disease was discovered to be a glycosphingolipidosis. Enzymes of lysosomal metabolism of glycosphingolipids were purified, characterized, and used in one of the first demonstrations of the feasibility of enzyme replacement therapy in a lysosomal storage disorder (Fabry’s disease). Extracellular sialidases were studied to evaluate the hypothesis that they might be involved in the regulation of membrane growth factor receptors. The enzyme for hematoside synthesis was purified and characterized.

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Peroxidase-amplified assay of sialidase activity toward gangliosides

Cited by 17 publications

References 18 publications

Calcium ions as a factor of cell—cell cooperation in hepatocyte cultures

Calcium ions as a factor of cell—cell cooperation in hepatocyte cultures

A Time-Resolved Immunofluorometric Assay of Sialyl Lewis x-Degrading α2,3-Sialidase Activity

Reflections on my career in analytical chemistry and biochemistry

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