Isolation of Chromaffin Granules

Carl E. Creutz1

1 Department of Pharmacology, University of Virginia, Charlottesville, Virginia
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.39
DOI:  10.1002/0471143030.cb0339s48
Online Posting Date:  September, 2010
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Abstract

Adrenal medullary chromaffin granules (dense core secretory vesicles) have been a valuable model system for the study of the proteins and membrane components involved in the process of exocytosis. Because of the abundance of chromaffin granules in a readily available tissue source, bovine adrenal medullae, and their unique sedimentation properties, it is possible to obtain large quantities of highly purified granules and granule membranes in a short period of time. Two protocols are presented here for the isolation of chromaffin granules: a basic protocol based on differential centrifugation in an iso‐osmotic medium that yields intact chromaffin granules, and an alternate protocol based on sedimentation through a density step gradient that provides a greater yield of more highly purified chromaffin granules. Since in the latter case the granules cannot be returned to a medium of physiological osmolarity without lysis after purification on the step gradient, the alternate protocol is more useful to obtain the granule membranes or contents for further study. Curr. Protoc. Cell Biol. 48:3.39.1‐3.39.10. © 2010 by John Wiley & Sons, Inc.

Keywords: chromaffin granule; chromaffin granule membrane; secretory vesicle; dense core vesicle; adrenal medulla; catecholamine; chromogranin; exocytosis; cytochrome b562; dopamine‐β‐hydroxylase; bovine

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Chromaffin Granules by Differential Centrifugation in Iso‐Osmotic Medium
  • Alternate Protocol 1: Isolation of Chromaffin Granules by Sedimentation Through a Sucrose Density Step
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation of Chromaffin Granules by Differential Centrifugation in Iso‐Osmotic Medium

  Materials
  • 5 to 30 bovine adrenal glands, freshly collected and transported to the laboratory
  • Cold 0.3 M sucrose solution (store at 4°C to inhibit bacterial growth), 1 liter
  • Surgical scissors, 2‐in. blades
  • 500‐ml beakers
  • Kitchen cutting board
  • Surgical scissors, 1‐in. blades
  • Mouse‐tooth forceps, 5‐in.
  • Waring blender
  • Potter‐Elvehjem glass homogenizer with a loose‐fitting Teflon pestle, 15 to 20 ml volume (a loose fitting pestle is one that shows little or no resistance or suction when moved up and down in the glass tube in air)
  • ¼‐in. electric hand drill for Potter‐Elvehjem pestle
  • Gauze sponges
  • Rubber band
  • 8‐in. stainless steel spatula with a ¼‐in. wide, rounded blade
  • Clear, 40‐ml polycarbonate centrifuge tubes
  • Refrigerated high speed centrifuge with Sorvall SS‐34 rotor or equivalent
  • 40‐ml Dounce homogenizer with loose‐fitting (type B) pestle
  • 15‐ml Dounce homogenizer with loose fitting (type B) pestle
NOTE: All glassware and equipment must be completely free of detergents in order to preserve the integrity of the chromaffin granule membranes.NOTE: After initial dissection of the adrenal glands, all materials are kept on ice.

Alternate Protocol 1: Isolation of Chromaffin Granules by Sedimentation Through a Sucrose Density Step

  • 250 ml cold 1.6 M sucrose solution, store at 4°C to inhibit bacterial growth
  • Clear, thick‐walled, 60‐ml polycarbonate ultracentrifuge tubes
  • 10‐ml pipet
  • Refrigerated ultracentrifuge and large‐capacity fixed‐angle rotor, e.g., Beckman Type 45 Ti
  • Tight‐fitting, type A pestle for the 40‐ml Dounce homogenizer
NOTE: All additional steps below are performed at 4°C or on ice. These steps must be undertaken immediately after the preparatory steps in the protocol 1 above; granules kept for hours or longer gradually lose their contents and will not sediment properly in a density gradient.
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Figures

Videos

Literature Cited

   Bartlett, S.F. and Smith, A.D. 1974. Adrenal chromaffin granules: Isolation and disassembly. Methods Enzymol. 31:379‐389.
   Creutz, C.E. 1981. Secretory vesicle‐cytosol interactions in exocytosis: Isolation by Ca2+‐dependent affinity chromatography of proteins that bind to the chromaffin granule membrane. Biochem. Biophys. Res. Commun. 103:1395‐1400.
   Creutz, C.E., Pazoles, C.J., and Pollard, H.B. 1978. Identification and purification of an adrenal medullary protein (synexin) that causes calcium‐dependent aggregation of isolated chromaffin granules. J. Biol. Chem. 253:2858‐2866.
   Creutz, C.E., Dowling, L.G., Sando, J.J., Villar‐Palasi, C., Whipple, J.H., and Zaks, W.J. 1983. Characterization of the chromobinins: Soluble proteins that bind to the chromaffin granule membrane in the presence of Ca2+. J. Biol. Chem. 258:14664‐14674.
   Douglas, W.W. and Poisner, A.M. 1966. Evidence that the secreting adrenal chromaffin cell releases catecholamines directly from ATP‐rich granules. J. Physiol. 183:236‐248.
   Fleming, P.J. and Kent, U.M. 1991. Cytochrome b561, ascorbic acid, and transmembrane electron transfer. Am. J. Clin. Nutr. 54:1174S‐1178S.
   Gratzl, M., Krieger‐Brauer, H., and Ekerdt, R. 1981. Latent acetylcholinesterase in secretory vesicles isolated from adrenal medulla. Biochim. Biophys. Acta 649:355‐366.
   Grynszpan‐Winograd, O. 1975. Ultrastructure of the chromaffin cell. In Handbook of Physiology, Section 7: Endocrinology, Volume VI: Adrenal Gland (H. Blaschko, G. Sayers, and A.D. Smith, eds.) pp. 295‐308. American Physiological Society, Bethesda, Md.
   Hillarp, N.‐Å. 1958. Isolation and some biochemical properties of the catechol amine granules in the cow adrenal medulla. Acta Physiol. Scand. 43:82‐96.
   Kirshner, N., Sage, H.J., Smith, W.J., and Kirshner, A.G. 1966. Release of catecholamines and specific protein from adrenal glands. Science 154:529‐531.
   Morris, S.J. and Schovanka, I. 1977. Some physical properties of adrenal medulla chromaffin granules isolated by a new continuous iso‐osmotic density gradient method. Biochim. Biophys. Acta 464:53‐64.
   Parsons, S.J. and Creutz, C.E. 1986. p60c‐src activity detected in the chromaffin granule membrane. Biochem. Biophys. Res. Commun. 134:736‐742.
   Pollard, H.B., Zinder, O., Hoffman, P.G., and Nikodejevic, O. 1976. Regulation of the transmembrane potential of isolated chromaffin granules by ATP, ATP analogs, and external pH. J. Biol. Chem. 251:4544‐4550.
   Pollard, H.B., Shindo, H., Creutz, C.E., Pazoles, C.J., and Cohen, J.S. 1979. Internal pH and state of ATP in adrenergic chromaffin granules determined by 31P nuclear magnetic resonance spectroscopy. J. Biol. Chem. 254:1170‐1177.
   Smith, A.D. and Winkler, H. 1967. A simple method for the isolation of adrenal chromaffin granules on a large scale. Biochem. J. 103:480‐482.
   Taugner, G. and Hasselbach, W. 1966. On the mechanism of catecholamine storage in the chromaffin granules of the adrenal medulla. Naunyn Schmiedebergs Arch. Exp. Pathol. Pharmakol. 255:266‐286.
   Trifaro, J.M. and Dworkind, J. 1970. A new and simple method for isolation of adrenal chromaffin granules by means of an isotonic density gradient. Anal. Biochem. 34:403‐412.
   Winkler, H., Apps, D.K., and Fischer‐Colbrie, R. 1986. The molecular function of adrenal chromaffin granules: Established facts and unresolved topics. Neuroscience 18:261‐290.
Key References
   Pollard, H.B., Pazoles, C.J., Creutz, C.E., and Zinder, O. 1979. The chromaffin granule and possible mechanisms of exocytosis. Int. Rev. Cytol. 58:159‐197.
  The above reviews provide a guide to the extensive literature on the chromaffin granule and the role it has played in the development of our understanding of secretory vesicle structure and function.
   Winkler et al., 1986. See above.
   Winkler, H. and Smith, A.D. 1975. The chromaffin granule and the storage of catecholamines. In Handbook of Physiology, Section 7: Endocrinology, Volume VI: Adrenal Gland (H. Blaschko, G. Sayers, and A.D. Smith, eds.) pp. 321‐339. American Physiological Society, Bethesda, MD.
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