Quantification of p38/synaptophysin in highly purified adrenal medullary chromaffin vesicles

Abstract

Chromaffin vesicles were first purified by differential and density gradient centrifugation in isotonic (Percoll) gradients. In subsequent sucrose gradients p38/synaptophysin exhibited the same distribution as established marker substances of chromaffin vesicles. Quantification of immunoblots revealed that 750 ng p38/synaptophysin per mg of protein were present in the chromaffin vesicles recovered from the sucrose gradient. Thus the amount of p38/synaptophysin per mg protein of chromaffin vesicles is about 100 times lower than that observed in clear (synaptic) vesicles. However, because of the large difference in surface area and protein content, the amount of p38/synaptophysin per single vesicle is the same in both types of organelles.p38/synaptophysin; Secretory vesicle; Immunoblotting; Quantification; (Adrenal medulla)

The Association of Dynamin with Synaptophysin Regulates Quantal Size and Duration of Exocytotic Events in Chromaffin Cells

“…In contrast, synaptophysin is broadly distributed across the sucrose density gradients. This result is consistent with synaptophysin being present not only in chromaffin granules (Obendorf et al, 1988;Schilling and Gratzl, 1988;Fournier et al, 1989) but also in other organelles such as synaptic-like microvesicles (Annaert et al, 1993), trans-Golgi networks (Régnier-Vigouroux et al, 1991), endosomes (Tixier-Vidal et al, 1988), and recycling vesicular organelles (Cameron et al, 1991;Linstedt and Kelly, 1991).…”

“…Being one of the most abundant integral proteins of synaptic vesicle membranes (Jahn et al, 1985), synaptophysin has been proposed to participate at several stages of the vesicle life cycle (Valtorta et al, 2004). This protein is also present in secretory granules of adrenal chromaffin cells (Obendorf et al, 1988;Schilling and Gratzl, 1988;Fournier et al, 1989) but at a lower density compared with synaptic vesicle membranes (Schilling and Gratzl, 1988).…”

Introduction of Macromolecules into Bovine Adrenal Medullary Chromaffin Cells and Rat Pheochromocytoma Cells (PC12) by Permeabilization with Streptolysin O: Inhibitory Effect of Tetanus Toxin on Catecholamine Secretion

“…The first was an antibody against vesicular protein p38, which is present in intracellular vesicles of endocrine and neuronal cells (Rehm et al, 1986;Navone et al, 1987;Schilling and Gratzl, 1988). The second was an antibody to calmodulin.…”

Fractionation of synaptophysin‐containing vesicles from rat brain and cultured PC12 pheochromocytoma cells

“…Synaptophysin is widely distributed in neoplastic neuroendocrine tissues and cell lines including the cell line PC12 derived from rat pheochromocytoma [3,10,12]. Immunoelectron- 14] recently provided evidence for the occurrence of synaptophysin in dense core granule membranes implying the participation of this polypeptide in at least two different secretory vesicle pathways. Because of this discrepancy between direct immunolocalization data and fractionation results, we now analyzed the synaptophysin content of vesicle fractions derived from rat and bovine brain, bovine adrenal medulla as well as cultured PC 12 cells by a newly developed dot immunoassay, which can detect 110 fmol synaptophysin.…”