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Solubilization and Characterization of Substance P Binding Protein from Bovine Brainstem
Author(s) -
Nakata Yoshihiro,
Tanaka Hiroyasu,
Morishima Yoshiyuki,
Segawa Tomio
Publication year - 1988
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.1471-4159.1988.tb02942.x
Subject(s) - chaps , stokes radius , binding protein , chemistry , chromatography , binding site , gtp' , fraction (chemistry) , size exclusion chromatography , biochemistry , solubilization , enzyme , gene
The specific binding protein for substance P (SP) was solubilized in an active form from the crude mitochondrial (P 2 ) fraction of bovine brainstem. After incubation with 3–[(3‐cholamidopropyl)dimethylammonio]‐1‐propane sulfonate (CHAPS) and 0.1 M NaCl at 0°C for 30 min, the SP binding to the supernatant fraction (100,000 g, 60 min) was determined by the glass fiber filtration method reported by Bruns et al. (1983). The specific [ 3 H]SP binding to the solubilized fraction was highly specific for SP and was displaced by nanomolar concentrations of SP and physalaemin, but only by micromolar concentrations of eledoisin. In addition, the binding was inhibited by GTP (approximately 40% of the specific binding decreased by 10 μ M GTP) in both preparations. These results were virtually identical to those of P 2 membrane preparations and suggested that this high‐affinity SP binding site belongs to the SP‐P type. Scatchard analyses of SP binding to the solubilized fraction revealed a single saturable component with a B max of 22.0 ± 5.10 fmol/mg protein and a K D of 0.79 n M , and these values are almost the same as those obtained in the P 2 fraction ( B max = 31.3 ± 3.56 fmol/mg protein, K D = 0.82 n M ). Gel filtration analysis showed that the detergent‐SP binding protein complex has two calculated molecular weights of > 1,000,000 and 55,000–60,000 (a corresponding Stokes radius of 35.5 nm).