Chapter 7: Microsomes
7.4 Chromaffin granules from adrenal glands
Figure 7.5 Schematic membrane of chromaffin granule
Table 7.3 Composition of chromaffin granules
- 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
- Lowry protein assay
- Catecholamine assay
- TEM fixatives and embedding materials
- Dissect out the medulla of an adrenal gland, weigh and
- 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
- 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.
- Collect the second pellet (pink, granular) and wash by
resuspending in fresh sucrose and recentrifuging @8,650 xg as
- Resuspend the washed pellet to yield a final protein
concentration of 60-80 mg/ml (as measured by OD).
- 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.
- 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.
- Fractionate the centrifuged chromaffin granules into 1.0 ml
fractions. Measure the absorbance of each fraction at 650 nm.
- Measure the ATP content of each fraction using a
commercially available luciferase reaction.
- Plot the fraction number vs the concentration of ATP.
Measure catecholamines by spectrophotometric analysis of
adrenochrome formation at 480 nm, using norepinephrine as a
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,
Determine total protein content by the Lowry method.
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.
A schematic of a chromaffin granule membrane is presented in
Return to table of contents
Cell Biology Laboratory Manual