Immunological Isolation of Hepatocyte Plasma Membranes

The plasma membrane is an important primary site of hormone action. Within its structure are hormone receptors and hormone-sensitive enzymes such as adenylate cyclase (Pohl et al., 1969) and possibly the low-K,,, cyclic AMP phosphodiesterase (Thompson et al., 1973). It is well established that insulin lowers cyclic AMP concentrations in intact fatand liver-cells but direct effects of insulin in membrane preparations on the enzymes of cyclic AMP metabolism have been difficult to obtain and the results of such studies remain controversial. One problem may be that existing methods for the purification of plasma membranes involve long periods at low temperatures and often produce only a low yield. Luzio et al. (1976) described a novel method for isolating fatcell plasma membranes immunologically. This method offered the possibility of rapidly preparing membranes in physiological buffers and only exposed membranes to low temperatures (4°C) for a short period of time. The present paper reports the use of this method, with modifications, for the preparation of liver cell plasma membranes together with preliminary data on the activities of adenylate cyclase and low-K,,, cyclic AMP phosphodiesterase in these membranes. Isolated liver cells were prepared by a modification of the method of Berry & Friend (1969). The liver of a male Wistar rat (200g) was perfused in reverse with Ca2+-free Hanks buffer containing sodium salts of pyruvate, fumarate and glutamate, all at concentrations of 5 m ~ , for 20min and then for a further 20min with the same medium containing 0.06% (w/v) collagenase. After perfusion the liver was suspended in KrebsRinger bicarbonate medium (1.3 mM-CaZ+), pH 7.4, containing 2 % (w/v) bovine serum albumin and dispersed with a spatula. The cells were then filtered, washed and finally resuspended in this medium. The resulting cell suspension (approx. 700mg dry wt. of cells in 200ml of medium) was incubated with rabbit anti-(rat erythrocyte) serum (8ml) for 30min at 37°C. The cells were then washed with Krebs-Ringer bicarbonate medium (1.3 mMCa2+), pH7.4, containing 0.1 % (w/v) bovine serum albumin and homogenized in a volume of approx. 200ml, by using a Polytron (P. T. 10 type “OD”) instrument on no. 7 setting for 6 x 20s at 4°C. Immunoadsorbents and non-immune adsorbents were prepared by coupling the appropriate sheep immunoglobulin G (IgG) to aminocellulose as described by Luzio