PURIFICATION AND PH-DEPENDENT SECRETORY VESICLE MEMBRANE-BINDING OF CHROMOGRANIN-B

Chromogranins A and B have been suggested to play crucial roles in the sorting of vesicular matrix proteins into secretory vesicles during vesicle biogenesis. Chromogranin A (CGA), a high-capacity, low-affinity Ca2+-binding protein, is the major protein in the secretory vesicles, while chromogranin B (CG8) is present in the vesicle at a significantly lower concentration. Chromogranin B has not been purified in its native form so far, thus severely limiting detailed studies of this protein. In the present study, chromogranin B was purified to complete homogeneity in its native state from the secretory vesicle lysates of bovine adrenal chromagranin cells using several chromatographic and electrophoresis steps. Recently, several intravesicular matrix proteins including chromogranins A and B have been shown to interact with the vesicle membrane at the intravesicular pH of 5.5 and to be released at a near-physiological pH of 7.5. However, since the experiment was done with the total vesicle lysate proteins, it was not clear whether CGB bound to the vesicle membrane directly or not. Hence, the pH-dependent binding of CGB to the vesicle membrane was tested using purified CGB, and it was found that pure CGB directly bound to the vesicle membrane at the intravesicular pH of 5.5. However, unlike the vesicle membrane-bound CGA, which can be easily eluted by a change of pH in the elution buffer from 5.5 to 7.5, the change of pH from 5.5 to 7.5 was not enough to elute the vesicle membrane-bound CGB. Its elution required a combination of both the pH change and a high salt concentration (1 M KCI), indicating that CGB had a higher affinity for the vesicle membrane than CGA. The tighter interaction of CGB with the vesicle membrane was further confirmed by vesicle membrane-coupled column chromatography using the mixture of CGA and CGB, In this regard, it appears that the high affinity of CGB for the membrane compensates its low concentration in the vesicle to ensure its interaction with the membrane, and the pH-dependent membrane binding of CGB may reflect the critical roles chromogranins A and B are suggested to play during vesicle biogenesis.