Solubilization and characterization of B2 bradykinin receptors from cultured human fibroblasts.
Active B2 bradykinin (BK) receptors were solubilized in high yields from intact monolayers or particulate fractions of cultured human foreskin fibroblasts using 4 mM of the non-denaturing zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). Other detergents showed only minor (digitonin) or no (Triton X-100, n-octyl glucopyranosid) efficacy at all. The stability of CHAPS-solubilized BK binding activity was temperature dependent being reduced to 30% of initial binding after 3 days of storage at 4 degrees C. CHAPS extracts, however, retained BK binding activity for at least several days when they were stored at -20 degrees C in the presence of 10% glycerol. The pharmacological characterization gave a rank order of potency for unlabeled BK, BK agonists, and antagonists to compete with [3H]BK for specific binding very similar to that observed in intact fibroblasts. Association and dissociation kinetics demonstrated that the binding of [3H]BK to the soluble CHAPS extracts was time dependent and reversible. Scatchard analysis of equilibrium binding data exhibited saturable binding of a single class of high affinity BK-binding sites with a Kd of 1.68 +/- 0.8 nM. Gel filtration revealed an apparent molecular weight of 250,000 for the solubilized BK receptor complex in CHAPS extracts. The ability to solubilize the B2 BK receptor in an active and stable form should allow for its future purification and for the characterization of its chemical properties.
Faussner
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A
Kinderklinik im Dr. von Haunerschen Kinderspital, Universität München, Germany.
Heinz-Erian
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P
Klier
C
C
Roscher
A A
AA
D001920:Bradykinin / Q000378:metabolism*; D002478:Cells, Cultured; D002793:Cholic Acids; D002850:Chromatography, Gel; D003902:Detergents; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D018002:Receptors, Bradykinin; D017981:Receptors, Neurotransmitter / Q000737:chemistry / Q000302:isolation & purification*; D012995:Solubility
D002793:Cholic Acids; D003902:Detergents; D018002:Receptors, Bradykinin; D017981:Receptors, Neurotransmitter; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D001920:Bradykinin