Iodination of Liver Plasma Membranes Using Lactoperoxidase: Effects on Adenylate Cyclase Activity

Abstract

In recent years, studies of receptors for many polypeptide hormones have focused on adenylate cyclase activity and hormone binding sites in isolated cells and purified plasma membrane preparations. These studies have provided major insights regarding the physiology of hormone action. However, relatively little has been learned of the molecular properties of receptors or of the chemical mechanisms involved in hormone receptor interactions. The major obstacle to obtaining a more basic understanding of receptors is their membrane location. While some information has been obtained with solubilized hormone-sensitive adenylate cyclase preparations (1-3), receptors must ultimately be studied in their natural membranous environment.

The lactoperoxidase-catalyzed iodination of exposed tyrosines has been used to label protein components of plasma membranes of several types of cells (4-6). The surface specificity of this enzyme suggested that it could provide useful information regarding the spatial arrangement of receptor components within an intact membrane. This report demonstrates the effects of iodination of highly purified plasma membranes from rat liver on adenylate cyclase activity in the membranes.

Materials and Methods. Lactoperoxidase, B grade, estimated purity 60%, was obtained from Calbiochem. Carrier-free ¹²⁵I was from Amersham/Searle. Acrylamide, N'N'-methylenebisacrylamide, and N,N, N, N-tetramethylethylenediamine were obtained from Eastman.

Plasma membranes were prepared by a modification (7) of the procedure of Neville (8). Adenylate cyclase was assayed as described previously (7) except that [³²P]cAMP was separated from [³²P]ATP by the aluminum oxide method devised by Ramachandran (9). Protein was measured by the procedure of Lowry et al. (10).

For amino acid analysis, liver membranes, 2 mg of protein, were hydrolyzed for 24 h at 100° in 6 N HCl. Amino acids were then estimated using a Spinco 120C amino-acid analyzer.

Results. Iodination of liver plasma membranes with lactoperoxidase has not been reported previously.