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Comparison of the Effects of Ions and GTP on Substance P Binding to Membrane‐Bound and Solubilized Specific Sites
Author(s) -
Morishima Yoshiyuki,
Nakata Yoshihiro,
Segawa Tomio
Publication year - 1989
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1989.tb08534.x
Subject(s) - chaps , membrane , gtp' , chemistry , pertussis toxin , chromatography , binding site , biophysics , biochemistry , g protein , enzyme , receptor , biology
Mg 2+ increased but Na + and GTP decreased [ 3 H]substance P (SP) binding to rat cerebral cortical membranes and to 10 m M 3‐[(3‐cholamidopropyl)dimethyl‐ammonio]‐l‐propane sulfonate (CHAPS)‐solubilized membrane fraction. To determine the binding parameters that are modified by the cations and GTP, inhibition experiments of [ 3 H]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 m M MgCl 2 was attained with a two‐site model, corresponding to a “high‐affinity (H)” and a “low‐affinity (L)” state. By omitting MgCl 2 , or by addition of NaCl and GTP, the [ 3 H]SP specific binding was decreased, the H state disappeared, and the L state and a new “super‐low affinity (SL)” state were observed. The SP/ [ 3 HJSP 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 MgC1 2 , 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 [ 3 HJSP binding by unlabeled SP in the presence of MgCl 2 in the CHAPS‐solubilized fraction were best fitted to a one‐site model. The K D 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 MgCI 2 concentration to 1 m M , although the inhibition curves consistently fit the one‐site model, the K D values changed only slightly.