Subcellular Distribution of 65,000 Calmodulin‐Binding Protein (p65) and Synaptophysin (p38) in Adrenal Medulla

Abstract

Both neuronal and endocrine cells contain secretory vesicles that store and release neurotransmitters and peptides. Neuronal cells release their secretory material from both small synaptic vesicles and large dense-core vesicles (LDCVs), whereas endocrine cells release secretory products from LDCVs. Neuronal small synaptic vesicles are known to express three integral membrane proteins: 65,000 calmodulin-binding protein (65-CMBP) (p65), synaptophysin (p38), and SV2. A controversial question surrounding these three proteins is whether they are present in LDCV membranes of endocrine and neuronal cells. Sucrose density centrifugation of adrenal medulla was performed to study and compare the subcellular distribution of two of these small synaptic vesicle proteins (65-CMBP and synaptophysin). Subsequent immunoblotting and 125I-Protein A binding experiments performed on the fractions obtained from sucrose gradients showed that 65-CMBP was present in fractions corresponding to granule membranes and intact chromaffin granules. Similar immunoblotting and 125I-Protein A binding experiments with synaptophysin antibodies showed that this protein was also present in intact granules and granule membrane fractions. However, an additional membrane component, equilibrating near the upper portion of the sucrose gradient, also showed strong immunoreactivity with anti-synaptophysin and high 125I-Protein A binding activity. In addition, immunoblotting experiments on purified plasma and granule membranes demonstrated that 65-CMBP was a component of both membranes, whereas synaptophysin was only present in granule membranes. Thus, there appears to be a different subcellular localization between 65-CMBP and synaptophysin in the chromaffin cell.

Role of the Vesicular Chloride Transporter ClC-3 in Neuroendocrine Tissue

“…The fractions were analyzed by Western blotting using carboxypeptidase E and secretogranin I and II as markers for LDCVs, and synaptophysin as a marker for SLMVs. In chromaffin cells, synaptophysin is not limited to SLMVs (like in ␤ cells) (see below), but is also found on LDCVs (Fournier et al, 1989). In the resulting biphasic distribution of synaptophysin across the gradient, the synaptophysin peak in fractions 7-11 represents SLMVs.…”

NSF and SNAP are present on adrenal chromaffin granules

“…1C) which overlapped with the two DBH positive peaks, a-SNAP was also detected in a peak corresponding to intact granules (fraction 9). A larger proportion of c~-SNAP was distributed in a broad peak over the lighter fractions of the gradient (fractions 1 6) where many other cell organelles sediment [25,26]. The most significant result from the analysis of these gradients is that a dense fraction of intact chromaffin granules is well separated from synaptic-like vesicles and contains substantial amounts of the total NSF and a-SNAP.…”

“…To examine the relative distribution of NSF and c(-SNAP between organelles in adrenal chromaffin cells and in particular to ensure that the NSF and e~-SNAP in the granule membrane fraction did not result from contamination with synaptic-like vesicles, post-nuclear organelles were fractionated on a 0.3 2.0 M sucrose gradient. The distribution of organelle markers on such gradients has been well characterised for adrenal medulla and it has been established that intact chromaffin granules sediment to higher density than any other post-nuclear organelles [24][25][26]. Fig.…”

Doc2 is not associated with known regulated exocytotic or endosomal compartments in adrenal chromaffin cells

“…The density of the Doc2-containing organelles on sucrose gradients also excludes the presence of Doc2 on Golgi compartments (cis-Golgi and trans-Golgi network), which run in much lighter fractions near the top of the gradients used for the analysis of intact postnuclear organelles (see also [22,23]). …”