Solubilization and reprecipitation from intestinal brush border membranes of a complex containing guanylate cyclase activatable by the heat-stable enterotoxin.
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 mM Hepes buffer (pH 7.4) and kept at 4 degrees C overnight, a precipitate formed. Centrifugation yielded a pellet (P2) 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 (P4). The addition of MgCl2 to the diluted extract affected both basal and STa-stimulated activity of P2; 1 mM was optimal. P2 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 P2 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 gamma-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 P2 and P4, 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. P2 and P4 appear to contain a complex of membrane proteins with certain functional properties intact.
John M. Dalton Research Center, University of Missouri-Columbia 65211.
L C
LC
Katwa
C D
CD
Parker
J K
JK
Dybing
A A
AA
White
D000246:Adenosine Diphosphate Ribose; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000494:pharmacology*; D002384:Catalysis; D011232:Chemical Precipitation; D002793:Cholic Acids; D003432:Cross-Linking Reagents; D003902:Detergents; D004768:Enterotoxins / Q000737:chemistry / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D029968:Escherichia coli Proteins; D006162:Guanylate Cyclase / Q000737:chemistry / Q000302:isolation & purification*; D006358:Hot Temperature; D007413:Intestinal Mucosa / Q000201:enzymology* / Q000648:ultrastructure; D008297:Male; D008871:Microvilli / Q000201:enzymology; D008970:Molecular Weight; D037342:Pertussis Toxin; D011189:Potassium Chloride / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000187:drug effects; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide; D012995:Solubility; D013552:Swine; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D001427:Bacterial Toxins; D002793:Cholic Acids; D003432:Cross-Linking Reagents; D003902:Detergents; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; D010566:Virulence Factors, Bordetella; C034151:heat stable toxin (E coli); D000246:Adenosine Diphosphate Ribose; D011189:Potassium Chloride; D037342:Pertussis Toxin; D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate