Chapter 7: Microsomes

7.4 Chromaffin granules from adrenal glands

LEVEL II

Figure 7.5 Schematic membrane of chromaffin granule

MATERIALS

• Bovine adrenal glands (preferred fresh from slaughterhouse)
  
• 0.27 M Sucrose, 10 mM Tris-maleate buffer, pH 7.0
  
• 30% Percoll (v/v) in 0.27 M sucrose, 10 mM Tris-maleate, pH 7.0
  
• Refrigerated, preparative centrifuge with swinging bucket rotor for 50 ml tubes
  
• Fraction collecting device
  
• ATP-luciferase kit 8
  
• UV/VIS spectrophotometer
  
• Optional
  
  • Lowry protein assay
    
  • Catecholamine assay
    
  • TEM fixatives and embedding materials

PROCEDURES 9

1. Dissect out the medulla of an adrenal gland, weigh and quickly mince.

2. For each gram of tissue, add 5 volumes of chilled 0.27 M sucrose, 10 mM Tris-maleate buffer, pH 7.0 and homogenize in a Potter-Elvijem homogenizer.

3. Centrifuge the brei for 5 minutes @ 1,250 xg @ 4 ° C . Discard the pellet and recentrifuge the supernatant @8,650 xg @4 ° C for 35 minutes.

4. Collect the second pellet (pink, granular) and wash by resuspending in fresh sucrose and recentrifuging @8,650 xg as above.

5. Resuspend the washed pellet to yield a final protein concentration of 60-80 mg/ml (as measured by OD).

6. Add 38 ml of 30% Percoll-sucrose in tris-maleate buffer to a 50 ml clear polycarbonate centrifuge tube and centrifuge the Percoll for 5 minutes @20,200 xg to form a gradient.

7. Add 2.0 ml of the resuspended chromaffin granule pellet to the top of the preformed Percoll gradient and centrifuge for 40 minutes @ 8,700 xg @ 4 ° C.
8. Fractionate the centrifuged chromaffin granules into 1.0 ml fractions. Measure the absorbance of each fraction at 650 nm.

9. Measure the ATP content of each fraction using a commercially available luciferase reaction.

10. Plot the fraction number vs the concentration of ATP.

OPTIONAL

Measure catecholamines by spectrophotometric analysis of adrenochrome formation at 480 nm, using norepinephrine as a standard. 10 Repellet the chromaffin granules and prepare a sample for electron microscopic observation. Monitor mitochondrial and lysosomal contamination via the presence of cytochrome oxidase and acid phosphatase, respectively. Determine total protein content by the Lowry method.

NOTES

Adrenal medullary tissue consists of cells containing chromaffin granules (named for their ability to stain with chromium stains) which are 100-300 nm in diameter. These granules consist of the catecholamine hormones epinephrine and norepinephrine (20% by weight), ATP (15%), protein (35%) and lipid (20%). About 85% of the granules store epinephrine, while the remainder store norepinephrine. Table 7.3 presents a more detailed chemical composition of chromaffin granules.

The adrenal medulla from cows and pigs has long served as a source of material for the isolation of specialized organelles which were originally believed to be storage vessicles for the catecholamines. These organelles are known as chromaffin granules and are embryologically related to the adrenergic neurotransmittor granules of the central nervous system. Lately, the presence of a proton pump has been identified within these granules and extensive studies are underway to relate the chemiosmotic concepts of membrane transport to the synthesis and release of the biologically active catecholamines. 11

A schematic of a chromaffin granule membrane is presented in figure 7.5.

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