Summary
A Percoll density gradient was employed for selecting large numbers of viable insulin-producing RINm5F cells. Homogenates of these cells were then subjected to gradient centrifugation and two clearly visible bands were obtained. The light fraction was essentially composed of mitochondria banded at a density of about 1.06 g/ml. The heavier fraction banded at 1.09 to 1.10 g/ml and contained lysosomes and a small number of secretory granules. The distribution of Percoll particles was restricted to the extracellular space and there was no adsorption to any membrane structures. The distribution pattern of marker enzymes for the mitochondria and lysosomes was similar to that of normal pancreatic β-cells. With the use of a Percoll density gradient it was thus possible to isolate a purified mitochondrial fraction from viable RINm5F cells.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Andersson, T.; Abrahamsson, H. Purification of mitochondria and secretory granules isolated from pancreatic β-cells using Percoll and Sephacryl S-1000 Superfine. Anal. Biochem. 132:82–88; 1983.
Bhatena, S. J.; Oie, H. K.; Gazdar, A. F.; et al. Insulin, glucagon, and somatostatin receptors on cultured cells and clones from rat islet cell tumor. Diabetes 31:521–531; 1982.
Carty, S. E.; Johnson, R.; Scarpa, A. The isolation of intact adrenal chromaffin granules using isotonic Percoll density gradients. Anal. Biochem. 106:438–445; 1980.
Chick, W. L.; Warren, S.; Chute, R. N.; et al. A transplantable insulinoma in the rat. Proc. Natl. Acad. Sci. USA 74:628–632; 1977.
Coore, H. G.; Denton, R. M.; Martin, B. R.; et al. Regulation of adipose tissue pyruvate dehydrogenase by insulin and other hormones. Biochem. J. 125:115–127; 1971.
Gazdar, A. F.; Chick, W. L.; Oie, H. K.; et al. Continuous, clonal, insulin-and somatostatin-secreting cell lines established from a transplantable rat islet cell tumor. Proc. Natl. Acad. Sci. USA 77:3519–3523; 1980.
Graham, J. Isolation of subcellular organelles and membranes. In: Rickwood, D. ed. Centrifugation—a practical approach, 2nd ed. Oxford: IRL Press; 1984.
Gylfe, E.; Andersson, T.; Rorsman, P.; et al. Depolarization-independent net uptake of calcium into clonal insulin-releasing cells exposed to glucose. Biosci. Rep. 3:927–937; 1983.
Heding, L. G. A simplified insulin radioimmunoassay method. In: Donato, L.; Milhaud, G.; Sirchis, I., eds. Labelled proteins in tracer studies. Brussels: European Atomic Energy Community; 1966:345–350.
Hutton, J. C.; Peshavaria, M. Proton-translocating Mg2+-dependent ATPase activity in insulin secretory granules. Biochem. J. 204:161–170; 1982.
Lagercrantz, H.; Pertoft, H.; Stjärne, L. Facts and artifacts in gradient centrifugation analysis of catecholamine granules. Acta Physiol. Scand. 78:561–566; 1970.
Meda, P. Lysosomes in normal pancreatic beta cells. Diabetologia 14:305–310; 1978.
Miller, G. L.; Gasek, J.; McGurren, J. Drift of drops in density gradient columns. Anal. Biochem. 1:78–87; 1960.
Oie, H.; Gazdar, A. F.; Minna, J. D.; et al. Clonal analysis of insulin and somatostatin secretion andl-dopa decarboxylase expression by a rat islet cell tumor. Endocrinology 112:1070–1075; 1983.
Percoll—Methodology and Applications. AB; Uppsala, Sweden: Pharmacia Fine Chemicals. 1982.
Pertoft, H.; Laurent, T. Isopycnic separation of cells by centrifugation in Percoll gradients. In: Cell function and differentiation, part A. New York: Alan R. Liss, Inc.; 1982:95–104.
Pertoft, H.; Wärmegård, B.; Höök, M. Heterogeneity of lysosomes originating from rat liver parenchymal cells—Metabolic relationship of subpopulations separated by density-gradient centrifugation. Biochem. J. 174:309–317; 1978.
Praz, G. A.; Halban, P. A.; Wollheim, C. B.; et al. Regulation of immunoreactive-insulin release from a rat cell line (RINm5F). Biochem. J. 210:345–352; 1983.
Reinhart, P.; Taylor, M.; Bygrave, F. A procedure for the rapid preparation of mitochondria from rat liver. Biochem. J. 204:731–735; 1982.
Rorsman, P.; Berggren, P.-O.; Gylfe, E.; et al. Reduction of cytosolic calcium activity in clonal insulin-releasing cells exposed to glucose. Biosci. Rep. 3:939–946; 1983.
Seglen, P. Separation approaches for liver and other cell sources. In: Reid, E., ed. Cell populations. Chichester, West Sussex, UK. Ellis Horwood Ltd.; 1979.
Sherman, W. R.; Stanfiled, E. F. Measurement of arylsulphatase ofPatella vulgata with 4-methylumbelliferone sulphate. Biochem. J. 102:905–909; 1967.
Wehrle, J.; Pedersen, P. Isolation and characterization of mitochondria from maligant cells. In: Reid, E., ed. Cancer-cell organelles. Chichester, West Sussex, UK. Ellis Horwood Ltd.; 1982.
Author information
Authors and Affiliations
Additional information
This work was supported by the Swedish Medical Research Council (03x-4, 12x-562, 12x-6240), the Swedish Diabetes Association, the Nordic Insulin Foundation, Syskonen Svenssons Foundation, and Åke Wiberg’s Foundation. Per-Olof Berggren is a recipient of a postdoctoral fellowship from the Swedish Medical Research Council.
Rights and permissions
About this article
Cite this article
Abrahamsson, H., Anderson, T., Berggren, PO. et al. Isolation of a purified mitochondrial fraction from viable clonal insulin-producing cells (RINm5F) by percoll density gradient centrifugation. In Vitro Cell Dev Biol 21, 415–420 (1985). https://doi.org/10.1007/BF02623473
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02623473