Molecular Pharmacology, Vol 8, 159-169, Copyright © 1972 by the American Society for Pharmacology and Experimental Therapeutics

Membranes of Adrenal Chromaffin Granules

Solubilization and Partial Characterization of the Mg⁺⁺-Dependent Adenosine Triphosphatase

¹ Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada

Membrane proteins were solubilized upon treatment of membranes obtained from adrenal medullary chromaffin granules with detergents (0.1-0.5% sodium dodecyl sulfate, 0.1% sodium deoxycholate, 0.02% Triton X-100, and 0.2-2.0% Lubrol PX). All solubilizing agents except Lubrol inactivated the chromaffin granule ATPase. Lubrol not only solubilize the membrane ATPase, but also increased the specific activity of the preparation 2.5 times.

Lubrol-solubilizod membrane protein were separated by gel filtration and by ion-exchange chromatography. The elution pattern of the granule ATPase changed with the Lubrol to protein ratio used during solubilization. With low ratios (4:1), the ATPase was eluted with the void volume from a Sephadex G-200 column, and immediately after the void volume from a Sepharose 6B column (K_av = 0.037), whereas when a 15:1 ratio was used the ATPase had a larger partition coefficient (K_av = 0.383).

Chromatography on DEAE-Sephadex A-25 showed that a fraction containing the ATPase activity was eluted at the ionic strength of 0.2, and that in the presence of Mg⁺⁺ transphosphorylation from ATP to granule protein was observed only in this fraction. The specific activity of the ATPase and the transphosphorylation from ATP in this fraction were 8-15 times higher than when tested in intact granule membranes.

Note:
ACKNOWLEDGMENTS We thank Mr. G. Duranceau of Legrade Abbatoir for providing us with the beef adrenals, and Mr. W. Mark for technical assistance. We are also grateful to Drs. B. Collier and A. Tenenhouse for reading the manuscript.