Comparison of the effects of ions and GTP on substance P binding to membrane-bound and solubilized specific sites.
Mg2+ increased but Na+ and GTP decrease [3H]substance P (SP) binding to rat cerebral cortical membranes and to 10 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS)-solubilized membrane fraction. To determine the binding parameters that are modified by the cations and GTP, inhibition experiments of [3H]SP binding by unlabeled SP were performed in both of the preparations. Nonlinear least-squares regression analysis of data in the membrane fraction indicated that optimal fitting of the inhibition curves in the presence of 10 mM MgCl2 was attained with a two-site model, corresponding to a "high-affinity (H)" and a "low-affinity (L)" state. By omitting MgCl2, or by addition of NaCl and GTP, the [3H]SP specific binding was decreased, the H state disappeared, and the L state and a new "super-low affinity (SL)" state observed. The SP/[3H]SP inhibition curves in the cerebral cortical membranes by in vivo treatment with pertussis toxin (islet-activating protein) were similar to that in the presence of GTP in control membranes. The effects of MgCl2, NaCl, and GTP were greater in the CHAPS-solubilized fraction than in the membrane fraction. In contrast to the membrane fraction, the inhibition curves of [3H]SP binding by unlabeled SP in the presence of MgCl2 in the CHAPS-solubilized fraction were best fitted to a one-site model. The KD value was relatively close to that of the low-affinity state in the membrane fraction. Even with the addition of NaCl or GTP, or by reducing MgCl2 concentration to 1 mM, although the inhibition curves consistently fit the one-site model, the KD values changed only slightly.
Morishima
Y
Y
Department of Pharmacology, Hiroshima University School of Medicine, Japan.
Nakata
Y
Y
Segawa
T
T
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000494:pharmacology*; D007477:Ions; D015636:Magnesium Chloride / Q000494:pharmacology; D008297:Male; D008566:Membranes / Q000378:metabolism; D037342:Pertussis Toxin; D051381:Rats; D011919:Rats, Inbred Strains; D012965:Sodium Chloride / Q000494:pharmacology; D012995:Solubility; D013373:Substance P / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D007477:Ions; D010566:Virulence Factors, Bordetella; D015636:Magnesium Chloride; D013373:Substance P; D012965:Sodium Chloride; D006160:Guanosine Triphosphate; D037342:Pertussis Toxin