Purification of Plasma Membranes from Rat Pancreas: A Rapid Method 1

Abstract

Introduction. The role of adenylate cy-clase and cyclic nucleotides in the secretory processes of the exocrine pancreas is not well defined. It has been reported that adenylate cyclase activity can be increased by secretin and, to a lesser degree, by pancreo-zymin ( 1 2 ). Kempen et al. ( 3 ) found that phospholipid addition increased the sensitivity of adenylate cyclase to pancreozymin. Moreover, some authors have reported that dibutyryl cAMP induces enzyme secretion ( 4 5 6 ), while others found no effect ( 7 8 ).

In spite of these controversial results, it is accepted that cyclic AMP could be involved as an intracellular mediator for hydralatic secretion ( 9 10 ), while cyclic GMP might be an intracellular messenger for enzyme secretion ( 11 12 ).

Purification of plasma membranes is a prerequisite for the study of molecular mechanisms underlying the hormonal and cholinergic responses of the exocrine pancreas. We describe here a rapid and simple method for the isolation of plasma membranes from the rat pancreas.

Materials and methods. ATP, GTP, AMP, yeast RNA, β-mercaptoethanol, Tris-ATP, creatine kinase, cAMP, phos-phocreatine, and albumin fraction V were obtained from Sigma. [α-³²P]ATP (150 Ci/ mmole) was purchased from New England Nuclear (Montreal); Metol was a product of Kodak. Synthetic terminal octapeptide of porcine cholecystokinin was a gift of Dr. M. A. Ondetti, Squibb Institute for Medical Research, Princeton, N. J. Natural porcine secretin was obtained from the GIH Research Unit, Karolinska Institutet, Stockholm, Sweden.

Adenylate cyclase assay. Adenylate cyclase activity was measured according to a modification ( 13 ) of the technique of Drum-mond and Duncan ( 14 ). The incubation was carried out for 15 min at 37° in a medium containing 40 μg/ml of soya bean trypsin inhibitor (SBTI), 0.02% albumin, 0.8 mM [α-³²P]ATP (4 μCi/assay), 10 mM theo-phylline, 5.5 mM MgCl₂, 40 mM Tris-HCl (pH 7.4), 50 μg of enzyme fraction protein, 100 μg of creatine kinase, and 20 mM phos-phocreatine in a final volume of 100 μI.