Solubilization and reprecipitation from intestinal brush border membranes of a complex containing guanylate cyclase activatable by the heat-stable enterotoxin

Abstract

Extraction of pig intestinal brush border membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (Chaps) in the presence of 0.5 m KCl yielded a solution which contained 60–70% of the receptor for the Escherichia coli heat-stable enterotoxin (STa) and of the Lubrol PX-activated guanylate cyclase activity present in the membrane. When the supernatant solution was diluted fivefold with 10 m m Hepes buffer (pH 7.4) and kept at 4 °C overnight, a precipitate formed. Centrifugation yielded a pellet (P 2) which contained 25–30% of both the cyclase and the receptor in the original membranes, with a 2.5- to 3-fold enrichment of both. The process could be repeated for further enrichment (P 4). The addition of MgCl 2 to the diluted extract affected both basal and STa-stimulated activity of P 2; 1 m m was optimal. P 2 resembled membranes with respect to competitive inhibition of 125I-STa binding by STa, and the concentration-dependent activation of cyclase by STa. Guanylate cyclase in resolubilized P 2 was also activated by STa. Most of the enzymes interfering with guanylate cyclase determinations were removed, as were the brush border marker enzymes sucrase and γ-glutamyltransferase, and a GTP-binding protein that is a pertussis toxin substrate. Specific cross-linking of 125I-STa to receptors in the membrane was preserved in P 2 and P 4, the three proteins showing the strongest radioactivity having relative molecular masses of 55,000–60,000, 70,000–80,000, and 135,000–140,000. P 2 and P 4 appear to contain a complex of membrane proteins with certain functional properties intact.