Isolation of Zymogen Granules from Rat Pancreas

Michael J. Rindler1

1 New York University School of Medicine, New York, New York
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.18
DOI:  10.1002/0471143030.cb0318s29
Online Posting Date:  January, 2006
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Abstract

This unit describes methods for preparing zymogen granules from rat pancreas. Zymogen granules are storage organelles in pancreatic acinar cells containing digestive enzymes that are released into the pancreatic duct. The protocols in this unit take advantage of the large size (up to 1 ┬Ám diameter) and high density (>1.20 g/cm3 on sucrose gradients) of the granules as compared to other cellular organelles. They use a combination of differential sedimentation and density gradient separation to accomplish the purification. Similar procedures can be used to isolate zymogen granules from mouse pancreas and canine pancreas. A protocol for preparing zymogen granules from dog pancreas is also included.

Keywords: pancreas; zymogen granules; secretory granules; amylase; exocrine; rat

     
 
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Table of Contents

  • Basic Protocol 1: Isolation of Zymogen Granules from Rat Pancreas
  • Support Protocol 1: Pouring a Continuous Sucrose Gradient
  • Support Protocol 2: Using a Refractometer
  • Alternate Protocol 1: Further Purification of Zymogen Granules on a Percoll Gradient
  • Alternate Protocol 2: Isolation of Zymogen Granule Content and Membrane Fractions
  • Alternate Protocol 3: Isolation of Zymogen Granules from Dog Pancreas
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Zymogen Granules from Rat Pancreas

  Materials
  • Rats (>200 g; Sprague‐Dawley)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Homogenization buffer (see recipe)
  • 30‐ml continuous sucrose gradients in 25 × 89–mm Beckman Ultraclear centrifuge tubes (0.8 to 2 M sucrose; see protocol 2)
  • Homogenization buffer (see recipe) containing 1 mM EDTA
  • CO 2 tank and vented chamber for inducing narcosis
  • Rat guillotine
  • Dissecting instruments including:
    • Dissecting scissors and small scissors
    • Scalpels or single‐edge razor blades
  • 150‐ and 250‐ml beakers
  • Glass plates, clean
  • Balance accurate to 0.1 g
  • 30‐ml piston‐type glass homogenizer‐tissue grinder with Teflon pestle (Thomas) powered by variable‐speed rotary drill (Wheaton Instruments)
  • 15‐ and 50‐ml conical polypropylene screw‐cap centrifuge tubes
  • Eppendorf 5810C centrifuge (or equivalent low‐speed refrigerated centrifuge) with swinging‐bucket rotor
  • 150‐ml Erlenmeyer flask
  • Nitex 20‐µm nylon mesh (Fisher)
  • 30‐ml glass Sorvall centrifuge tubes
  • Sorvall RC‐5B centrifuge with SS‐34 rotor (or equivalent)
  • 7‐ml Dounce homogenizer with B pestle (Kontes Glass)
  • Optima L‐90K ultracentrifuge with SW‐28 rotor (Beckman Coulter) or equivalent ultracentrifuge with swinging‐bucket rotor
  • Additional reagents on equipment for preparing a sucrose gradient ( protocol 2) and using a refractometer ( protocol 3)
NOTE: Wear a laboratory coat and gloves throughout the procedure. All steps should be performed at 4°C, in a cold room if possible.

Support Protocol 1: Pouring a Continuous Sucrose Gradient

  Materials
  • 0.8 and 2 M sucrose gradient solutions (see recipe)
  • 39‐ml, 25 × 89–mm Beckman Ultraclear centrifuge tube
  • Auto Densi‐Flow device (Buchler Instruments)
  • Peristaltic pump (Buchler Instruments)
  • Gradient maker (Hoefer Instruments SG30)
  • Magnetic stirrer and stir bars
  • Parafilm

Support Protocol 2: Using a Refractometer

  Materials
  • Sucrose gradient fractions ( protocol 1)
  • Refractometer (Bausch & Lomb)

Alternate Protocol 1: Further Purification of Zymogen Granules on a Percoll Gradient

  • Crude zymogen granule fraction ( protocol 1, step )
  • Percoll gradient solution (see recipe)
  • 50 ml, 29 × 102–mm flanged polycarbonate tubes centrifuge tubes (Fisher)
  • Sorvall RC‐5B with SS‐34 rotor or equivalent centrifuge with fixed‐angle rotor

Alternate Protocol 2: Isolation of Zymogen Granule Content and Membrane Fractions

  • Percoll‐purified zymogen granules ( protocol 4)
  • Lysis buffer (see recipe)
  • 0.75 M and 0.95 M sucrose gradient solutions (see recipe)
  • 25 mM Tris·Cl, pH 7.5 containing 1 mM EDTA
  • 13.2 ml, 14 × 89–mm Ultraclear centrifuge tubes
  • Beckman ultracentrifuge with SW‐41 rotor (or equivalent)
  • 15‐ml conical polypropylene centrifuge tubes
  • Probe sonicator (Heat Systems Ultrasonics Model 185 or equivalent) with pencil probe
  • Beckman TL‐100 centrifuge and TLA 100.3 rotor (or equivalent)
  • 13 × 31–mm thick‐walled polycarbonate centrifuge tubes or polyallomer microcentrifuge tubes

Alternate Protocol 3: Isolation of Zymogen Granules from Dog Pancreas

  • Dog, newly sacrificed
  • 1‐liter beakers
  • Tissue press consisting of a piston that drives the tissue through a 1‐mm stainless steel mesh (a good‐quality garlic press may be substituted)
  • 250‐ml disposable plastic conical screw‐cap centrifuge tubes (Corning)
  • Sorvall RC‐5B centrifuge with GSA rotor (or equivalent refrigerated centrifuge with fixed‐angle rotor)
  • 40‐ml Dounce homogenizer with B pestle
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Figures

Videos

Literature Cited

Literature Cited
   Bonner‐Weir, S., Deery, D., Leahy, J.L., and Weir, G.C. 1989. Compensatory growth of pancreatic beta‐cells in adult rats after short‐term glucose infusion. Diabetes 38:49‐53.
   Cameron, R.S., Cameron, P.L., and Castle, J.D. 1986. A common spectrum of polypeptides occurs in secretion granule membranes of different exocrine glands. J. Cell Biol. 103:1299‐1313.
   De Lisle, R.C., Schulz, I., Tyrakowski, T., Haase, W., and Hopfer, U. 1984. Isolation of stable pancreatic zymogen granules. Am. J. Physiol. 246:G411‐G418.
   Greene, L.J., Hirs, C.H., and Palade, G.E. 1963. On the protein composition of bovine pancreatic zymogen granules. J. Biol. Chem. 238:2054‐2070.
   Hokin, L.E. 1955. Isolation of the zymogen granules of dog pancreas and a study of their properties. Biochim. Biophys. Acta 18:379‐388.
   Kleene, R., Dartsch, H., and Kern, H.F. 1999. The secretory lectin ZG16p mediates sorting of enzyme proteins to the zymogen granule membrane in pancreatic acinar cells. Eur. J. Cell Biol. 78:79‐90.
   Paquet, M.R., St‐Jean, P., Roberge, M., and Beaudoin, A.R. 1982. Isolation of zymogen granules from rat pancreas and characterization of their membrane proteins. Eur. J. Cell Biol. 28:20‐26.
   Pennington, R.J. 1961. Biochemistry of dystrophic muscle. Mitochondrial succinate‐tetrazolium reductase and adenosine triphosphatase. Biochem. J. 80:649‐654.
   Rhodes, C.J., Lucas, C.A., Mutkoski, R.L., Orci, L., and Halban, P.A. 1987. Stimulation by ATP of proinsulin to insulin conversion in isolated rat pancreatic islet secretory granules. Association with the ATP‐dependent proton pump. J. Biol. Chem. 262:10712‐10717.
   Rindler, M.J. 1992. Biogenesis of storage granules and vesicles. Curr. Opin. Cell Biol. 4:616‐622.
   Rindler, M.J. and Hoops, T.C. 1990. The pancreatic membrane protein GP‐2 localizes specifically to secretory granules and is shed into the pancreatic juice as a protein aggregate. Eur. J. Cell Biol. 53:154‐163.
   Scheele, G.A. 1981. Analysis of the secretory process in the exocrine pancreas by two‐dimensional isoelectric focusing/sodium dodecyl sulfate gel electrophoresis. Methods Cell Biol. 23:345‐358.
   Tartakoff, A., Greene, L.J., and Palade, G.E. 1974. Studies on the guinea pig pancreas. Fractionation and partial characterization of exocrine proteins. J. Biol. Chem. 249:7420‐7431.
   Wagner, A.C., Wishart, M.J., Mulders, S.M., Blevins, P.M., Andrews, P.C., Lowe, A.W., and Williams, J.A. 1994. GP‐3, a newly characterized glycoprotein on the inner surface of the zymogen granule membrane, undergoes regulated secretion. J. Biol. Chem. 269:9099‐9104.
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