Dense core secretory vesicles, represented by chromaffin secretory vesicles (also known as chromaffin granules), were purified from fresh bovine adrenal medulla by differential sucrose density gradient centrifugation, as described previously (37 ,38 (link)), involving extensive wash steps to obtain purified chromaffin granules. We have documented the high purity of this preparation of isolated secretory vesicles by electron microscopy (Fig. 1 ) and biochemical markers (36 (link)–38 (link)). Sucrose gradient purification results in a preparation of purified, intact chromaffin secretory vesicles that lack biochemical markers for the subcellular organelles of lysosomes (acid phosphatase marker) (38 (link)), cytoplasm (lactate dehydrogenase marker) (37 ), mitochondria (fumarase and glutamate dehydrogenase markers) (36 (link),37 ), and endoplasmic reticulum (glucose-6-phosphatase marker) (37 ). Enzyme markers have been measured in the purified chromaffin secretory vesicle preparation as 1% or less of total homogenate markers, which, thus, indicate the high purity of these isolated secretory vesicles (36 (link)–38 (link)).
In addition, this study further assessed the removal of the lysosomal enzyme marker acid phosphatase from the purified preparation of chromaffin granules compared to unpurified sample of chromaffin granules obtained at an early step in the purification procedure (illustrated infigure 1a ). Purified and unpurified granules were analyzed on a multi-step sucrose gradient of 2.2 to 1.2 M sucrose (2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, and 1.2 M sucrose steps each consisting of 2.5 ml) by ultracentrifugation at 120,000 × g in a SW28 rotor (25,000 rpm) at 4° C for 100 min. Gradient fractions of 0.5 ml were collected from the bottom of the tube (2.2 M sucrose), and fractions were assayed for (Met)enkephalin by RIA as previously described (5 (link)) as a marker for chromaffin granules, and acid phosphatase activity as a marker for lysosomes as described previously (38 (link)). Results show that the purified chromaffin granules lack acid phosphatase activity, indicating effective removal of lysosomes of density near that of chromaffin granules (explained in fig. 1 of results). These new data and established purity in the literature (36 (link)–38 (link)) document the purity of these chromaffin secretory vesicles for this study.
Soluble and membrane components of the purified chromaffin granules were prepared by lysing (by freeze-thawing) purified chromaffin granules in isotonic buffer conditions consisting of 150 mM NaCl in 50 mM Na-acetate, pH 6.0, with a cocktail of protease inhibitors (10 μM pepstatin A, 10 μM leupeptin, 10 μM chymostatin, 10 μM E64c, and 1 mM AEBSF). The lysed granules were centrifuged at 100,000 × g (SW60 rotor) at 4° C for 30 minutes. The resultant supernatant was collected as the soluble fraction. The pellet was collected as the membrane fraction, and washed two times by re-suspending in the lysis buffer and centrifugation (100,000 × g, 30 min). The final pellet was resuspended in the lysis buffer and designated as the membrane fraction.
The soluble and membrane fractions were each subjected to removal of the abundant chromogranin A (CgA) protein, by its binding to calmodulin-Sepharose (GE Healthcare, formerly Amersham Biosciences, Piscataway, NJ) (39 (link)). The soluble fraction and membrane fraction (solubilized in 50 mM CHAPS) were each incubated with a slurry of calmodulin-Sepharose at 4° C overnight in equilibration buffer (50 mM Tris-HCl, pH 7.5, 0.1 M NaCl, 2 mM CaCl2, and protease inhibitors consisting of 5 μM E64c, 5 μM leupeptin, 5 μM chymostatin, 5 μM pepstatin A, 5 μM bestatin, 1 μM GEMSA, and 50 mM PMSF). The mixture was centrifuged and the supernatant collected as the soluble fraction without CgA. This step removed approximately 90–95% of CgA, based on assessment by anti-CgA western blots.
Proteins in the membrane fraction were concentrated by chloroform-methanol precipitation. To the membrane fraction (400 μg in 300 μl) was added MeOH (400 μl), chloroform (100 μl), and deionized water (300 μl) with mixing between each step, followed by centrifugation (14,000 × g for 1 min). The top aqueous layer was removed, while retaining the protein precipitate at the top of the chloroform layer; after addition of MeOH (400 μl), mixing, and centrifugation (14,000 × g for 2 min), the pelleted protein was collected for trypsin digestion (40 (link)).
In addition, this study further assessed the removal of the lysosomal enzyme marker acid phosphatase from the purified preparation of chromaffin granules compared to unpurified sample of chromaffin granules obtained at an early step in the purification procedure (illustrated in
Soluble and membrane components of the purified chromaffin granules were prepared by lysing (by freeze-thawing) purified chromaffin granules in isotonic buffer conditions consisting of 150 mM NaCl in 50 mM Na-acetate, pH 6.0, with a cocktail of protease inhibitors (10 μM pepstatin A, 10 μM leupeptin, 10 μM chymostatin, 10 μM E64c, and 1 mM AEBSF). The lysed granules were centrifuged at 100,000 × g (SW60 rotor) at 4° C for 30 minutes. The resultant supernatant was collected as the soluble fraction. The pellet was collected as the membrane fraction, and washed two times by re-suspending in the lysis buffer and centrifugation (100,000 × g, 30 min). The final pellet was resuspended in the lysis buffer and designated as the membrane fraction.
The soluble and membrane fractions were each subjected to removal of the abundant chromogranin A (CgA) protein, by its binding to calmodulin-Sepharose (GE Healthcare, formerly Amersham Biosciences, Piscataway, NJ) (39 (link)). The soluble fraction and membrane fraction (solubilized in 50 mM CHAPS) were each incubated with a slurry of calmodulin-Sepharose at 4° C overnight in equilibration buffer (50 mM Tris-HCl, pH 7.5, 0.1 M NaCl, 2 mM CaCl2, and protease inhibitors consisting of 5 μM E64c, 5 μM leupeptin, 5 μM chymostatin, 5 μM pepstatin A, 5 μM bestatin, 1 μM GEMSA, and 50 mM PMSF). The mixture was centrifuged and the supernatant collected as the soluble fraction without CgA. This step removed approximately 90–95% of CgA, based on assessment by anti-CgA western blots.
Proteins in the membrane fraction were concentrated by chloroform-methanol precipitation. To the membrane fraction (400 μg in 300 μl) was added MeOH (400 μl), chloroform (100 μl), and deionized water (300 μl) with mixing between each step, followed by centrifugation (14,000 × g for 1 min). The top aqueous layer was removed, while retaining the protein precipitate at the top of the chloroform layer; after addition of MeOH (400 μl), mixing, and centrifugation (14,000 × g for 2 min), the pelleted protein was collected for trypsin digestion (40 (link)).
