Navegar

Colección

Datos Estadísticas

Artículo

Estamos trabajando para incorporar este artículo al repositorio
Consulte el artículo en la página del editor
Consulte la política de Acceso Abierto del editor

Abstract:

The paper shows the ability of the fluorochrome tris(2,2′-bipyridine) ruthenium (II) (Rubipy) to detect heparan sulfate, heparin, and heparinase activity of M3 murine mammary adenocarcinoma cells as well as bacterial heparinases I, II, and III in native polyacrylamide gel electrophoresis (PAGE). The technique is based on the electrophoretic mobility of high molecular weight heparins and subsequent staining with Rubipy (50 μg/mL). The minimum content of heparin detected by fluorescence in a UV transilluminator was 25-50 ng. The number of Rubipy molecules bound to heparin, determined in relationship to the number of disaccharide units (DU), showed that two to six heparin disaccharide units are bound by each fluorochrome molecule. Scatchard plot analysis showed one Rubipy-binding site (Kd= (8.56 ± 2.97) × 10-5 M). Heparinase activity was determined by densitometric analysis of the fluorescence intensity of the heparin-containing band of the gel. While heparinase I (EC 4.2.2.7.) degraded heparin and, to a lower degree, partially N-desulfated N-acetylated heparin (N-des N-Ac), heparinase II (no EC number) could efficiently degrade heparan sulfate (HS) and partially N-des N-Ac heparin. Finally, heparinase III (EC 4.2.2.8.) degraded HS almost exclusively. Only heparin and N-des N-Ac heparin were substrates for M3 tumor cell heparinases. We describe a qualitative, sensitive and simple method to detect heparinase activity and determine its substrate specificity using Rubipy fluorescence with heparin and heparan sulfate in multiple biological samples tested in parallel.

Registro:

Documento: Artículo
Título:Heparan sulfate, heparin, and heparinase activity detection on polyacrylamide gel electrophoresis using the fluorochrome tris(2,2′-bipyridine) ruthenium
Autor:Rozenberg, G.I.; Espada, J.; de Cidre, L.L.; Eiján, A.M.; Calvo, J.C.; Bertolesi, G.E.
Filiación:Departamento de Química Biológica, Argentina
Departamento de Ciencas Biológicas, Universidad de Buenos Aires, Argentina
Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Madrid, Spain
Instituto de Oncología 'A.H.Roffo', Universidad de Buenos Aires, Argentina
Departamento de Química Biológica, Universidad de Buenos Aires, Argentina
Palabras clave:Heparan sutfate; Heparin; Heparinase; Tris (2,2′bipyridine) ruthenium (II) EL 4139; 2,2' bipyridine; fluorochrome; heparan sulfate; heparin; heparin lyase; ruthenium complex; tris(2,2' bipyridine)ruthenium; unclassified drug; adenocarcinoma; analytic method; animal cell; article; binding site; carcinoma cell; densitometry; electrophoretic mobility; enzyme activity; enzyme specificity; fluorescence; nonhuman; polyacrylamide gel electrophoresis; 2,2'-Dipyridyl; Adenocarcinoma; Animals; Electrophoresis, Polyacrylamide Gel; Female; Fluorescent Dyes; Heparin; Heparin Lyase; Heparitin Sulfate; Mammary Neoplasms, Animal; Mice; Molecular Structure; Polysaccharide-Lyases; Tumor Cells, Cultured
Año:2001
Volumen:22
Número:1
Página de inicio:3
Página de fin:11
DOI: http://dx.doi.org/10.1002/1522-2683(200101)22:1<3::AID-ELPS3>3.0.CO;2-G
Título revista:Electrophoresis
Título revista abreviado:Electrophoresis
ISSN:01730835
CODEN:ELCTD
CAS:2,2' bipyridine, 366-18-7; fluorochrome, 37332-03-9; heparan sulfate, 9050-30-0; heparin, 37187-54-5, 8057-48-5, 8065-01-8, 9005-48-5; heparin lyase, 37290-85-0, 9025-39-2; tris(2,2' bipyridine)ruthenium, 14323-06-9; 2,2'-Dipyridyl, 366-18-7; Fluorescent Dyes; Heparin, 9005-49-6; Heparin Lyase, 4.2.2.7; Heparitin Sulfate, 9050-30-0; Polysaccharide-Lyases, 4.2.2.-; heparinase II, 4.2.2.-; heparitinsulfate lyase, 4.2.2.8; tris(2,2'-bipyridine)ruthenium II, 15158-62-0
Registro:https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_01730835_v22_n1_p3_Rozenberg

Referencias:

  • Casu, B., (1985) Adv. Carbohydr. Chem. Biochem., 43, pp. 51-134
  • Linhardt, R.J., Turnbull, J.E., Wang, H.M., Loganathan, D., Gallagher, J.T., (1990) Biochem., 29, pp. 2611-2617
  • Jandik, K.A., Gu, K., Linhardt, R.J., (1994) Glycobiol., 4, pp. 289-296
  • Matzner, Y., Bar-Ner, M., Yahalom, J., Ishai-Michaeli, R., Fuks, Z., Voldavsky, I., (1985) J. Clin. Invest., 76, pp. 1306-1313
  • Bashkin, P., Razin, E., Eldor, A., Vlodavsky, I., (1990) Blood, 75, pp. 2204-2212
  • Vlodavsky, I., Eldor, A., Hairnovitz-Friedman, A., Matzner, Y., Ishai-Michaeli, R., Lider, O., Naparstek, Y., Fuks, Z., (1992) J. Invas. Metast., 12, pp. 112-127
  • Nakajima, M., Irimura, T., Nicolson, G.L., (1988) J. Cell. Biochem., 36, pp. 157-167
  • Jin, L., Nakajima, M., Nicolson, G.L., (1990) Int. J. Cancer, 45, pp. 1088-1095
  • Vlodavsky, I., Friedmann, Y., Elkin, M., Aingorn, H., Atzmon, R., Ishai-Michaeli, R., Bian, M., Pecker, I., (1999) Nature Med., 5, pp. 793-802
  • Hulett, M.D., Freeman, C., Hamdorf, B.J., Baker, R.T., Harris, M.J., Parish, C.R., (1999) Nature Med., 5, pp. 803-809
  • Nakajima, M., Irimura, T., Di Ferrante, D., Di Ferrante, N., Nicolson, G.L., (1983) Science, 220, pp. 611-613
  • De Vouge, M.W., Yamazaki, A., Bennett, S.A., Chen, J.H., Shwed, P.S., Couture, C., Birnboim, H.C., (1994) Int. J. Cancer, 56, pp. 286-294
  • Al-Hakim, A., Linhardt, R.J., (1991) Appl. Theor. Electrophor., 1, pp. 305-312
  • Linker, A., Hovingh, P., (1972) Biochemistry, 11, pp. 563-568
  • Bertolesi, G.E., Trigoso, C.I., Espada, J., Stockert, J.C., (1995) J. Histochem. Cytochem., 43, pp. 537-543
  • Bertolesi, G.E., Stockert, J.C., Lauría de Cidre, L., (1997) Int. J. Oncol., 11, pp. 1221-1225
  • Kelly, J.M., Tossi, A.M., McConnell, D.J., OhUigin, C., (1985) Nucleic Acids Res., 13, pp. 6017-6034
  • Bitter, T., Muir, H.M., (1962) Anal. Biochem., 4, pp. 330-334
  • Laemmli, U.K., (1970) Nature, 227, pp. 680-685
  • Munson, P.J., Rodbard, D., (1980) Anal. Biochem., 107, pp. 220-239
  • Bal de Kier Joffé, E., Puricelli, L., Vidal, M.C., Sacerdote de Lustig, E., (1983) J. Exp. Clin. Cancer Res., 2, pp. 151-160
  • Bal de Kier Joffé, E., Puricelli, L., Sacerdote de Lustig, E., (1986) Invasion Metastasis, 6, pp. 302-312
  • Johnson, E.A., Mulloy, B., (1976) Carbohydr. Res., 51, pp. 119-127
  • Tas, J., Roozemond, R.C., (1973) Histochem. J., 5, pp. 425-436
  • Stone, A.L., Bradley, D.F., (1967) Biochim Biophys. Acta, 148, pp. 172-192
  • Desai, U.R., Wang, H., Ampofo, S.A., Linhardt, R.J., (1993) Anal. Biochem., 213, pp. 120-127
  • Rusnati, M., Tulipano, G., Spillman, D., Tanghetti, E., Oreste, P., Zoppetti, G., Gliacca, M., Presta, M.J., (1999) Biol. Chem., 274, pp. 28198-28205
  • Nader, H.B., Porcionatto, M.A., Tersariol, I.L.S., Pinhal, M.A.S., Oliveira, F.W., Moraes, C.T., Dietrich, C.P., (1990) J. Biol. Chem., 265, pp. 16807-16813
  • Sasisekharan, R., Bulmer, M., Moremen, K.W., Cooney, C.L., Langer, R., (1993) Proc. Natl. Acad. Sci. USA, 90, pp. 3660-3664
  • Bar-Ner, M., Eldor, A., Wasserman, L., Matzner, Y., Cohen, I.R., Fuks, Z., Vlodavsky, I., (1987) Blood, 70, pp. 551-557
  • Bertolesi, G.E., Lauría de Cidre, L., Sacerdote de Lustig, E., Eiján, A.M., (1995) Cancer Lett., 90, pp. 123-131
  • Brzu, T., Pascal, M., Maman, M., Roque, C., Lafont, F., Rousselet, A., (1996) J. Cell. Physiol., 167, pp. 8-21
  • Khan, M.Y., Newman, S.A., (1991) Anal. Biochem., 196, pp. 373-376

Citas:

---------- APA ----------
Rozenberg, G.I., Espada, J., de Cidre, L.L., Eiján, A.M., Calvo, J.C. & Bertolesi, G.E. (2001) . Heparan sulfate, heparin, and heparinase activity detection on polyacrylamide gel electrophoresis using the fluorochrome tris(2,2′-bipyridine) ruthenium. Electrophoresis, 22(1), 3-11.
http://dx.doi.org/10.1002/1522-2683(200101)22:1<3::AID-ELPS3>3.0.CO;2-G
---------- CHICAGO ----------
Rozenberg, G.I., Espada, J., de Cidre, L.L., Eiján, A.M., Calvo, J.C., Bertolesi, G.E. "Heparan sulfate, heparin, and heparinase activity detection on polyacrylamide gel electrophoresis using the fluorochrome tris(2,2′-bipyridine) ruthenium" . Electrophoresis 22, no. 1 (2001) : 3-11.
http://dx.doi.org/10.1002/1522-2683(200101)22:1<3::AID-ELPS3>3.0.CO;2-G
---------- MLA ----------
Rozenberg, G.I., Espada, J., de Cidre, L.L., Eiján, A.M., Calvo, J.C., Bertolesi, G.E. "Heparan sulfate, heparin, and heparinase activity detection on polyacrylamide gel electrophoresis using the fluorochrome tris(2,2′-bipyridine) ruthenium" . Electrophoresis, vol. 22, no. 1, 2001, pp. 3-11.
http://dx.doi.org/10.1002/1522-2683(200101)22:1<3::AID-ELPS3>3.0.CO;2-G
---------- VANCOUVER ----------
Rozenberg, G.I., Espada, J., de Cidre, L.L., Eiján, A.M., Calvo, J.C., Bertolesi, G.E. Heparan sulfate, heparin, and heparinase activity detection on polyacrylamide gel electrophoresis using the fluorochrome tris(2,2′-bipyridine) ruthenium. Electrophoresis. 2001;22(1):3-11.
http://dx.doi.org/10.1002/1522-2683(200101)22:1<3::AID-ELPS3>3.0.CO;2-G