Solubilization and characterization of functionally coupled Escherichia coli heat-stable toxin receptors and particulate guanylate cyclase associated with the cytoskeleton compartment of intestinal membranes.
1. Particulate guanylate cyclase and receptors for E. coli heat-stable enterotoxin were solubilized from the rat intestinal cytoskeletal compartment using Lubrol-PX and KCl. 2. Thirty to forty percent of the ST receptor and guanylate cyclase activities were extracted from the lipid layer with Lubrol-PX alone. 2. Seventy percent of the remaining activities were solubilized from the cytoskeleton with Lubrol-PX and KCl. 3. Guanylate cyclase solubilized from either compartment exhibited similar reaction kinetics. 4. Both high- and low-affinity classes of ST receptors were solubilized from the lipid and cytoskeleton compartments. 5. In the presence of ATP gamma S, ST selectively activated the guanylate cyclase solubilized from the cytoskeleton compared to that solubilized from the lipid bilayer. 6. Crosslinking experiments demonstrated a preferential solubilization of the 130 kDa receptor subunit from the cytoskeleton and the 56 kDa subunit from the lipid bilayer. 7. Development of a procedure to solubilize ST receptors and guanylate cyclase from the intestinal membrane cytoskeleton will permit purification and further detailed studies of the coupling of these activities.
Hakki
S
S
Department of Medicine, Thomas Jefferson University, Philadelphia, PA 19107.
Crane
M
M
Hugues
M
M
O'Hanley
P
P
Waldman
S A
SA
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism*; D002462:Cell Membrane / Q000648:ultrastructure; D003599:Cytoskeleton / Q000737:chemistry*; D004768:Enterotoxins / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D006162:Guanylate Cyclase / Q000737:chemistry / Q000302:isolation & purification*; D007422:Intestines / Q000648:ultrastructure*; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry; D000077423:Polidocanol; D011092:Polyethylene Glycols; D011189:Potassium Chloride; D051381:Rats; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide; D012995:Solubility
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; C034151:heat stable toxin (E coli); D000077423:Polidocanol; C022571:adenosine 5'-O-(3-thiotriphosphate); D011092:Polyethylene Glycols; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled