Purification of proctolin-binding proteins from the foregut of the insect Blaberus craniifer.
A membrane protein that specifically binds the insect neuropeptide proctolin was purified using standard chromatography from cockroach foregut membranes. Proctolin-binding sites were efficiently solubilized with either the nonionic detergent digitonin or the zwitterionic detergent Chaps, as indicated by the specific binding of 3H-proctolin to solubilized samples. A solubilized sample obtained from 1600 foregut membranes was subjected to a five-step chromatographic purification including chromatofocusing, anion-exchange and size-exclusion chromatographies. The final size-exclusion separation resulted in the isolation of approximately 100 pmol of purified proctolin-binding proteins, eluting as a single peak at approximately 74 kDa. Analysis of the purified sample using SDS/PAGE and silver staining showed two bands at 80 kDa and 76 kDa. Densitometric analysis of the gel indicated that each band contained approximately 7-8 microg of protein, suggesting that one band corresponds to the proctolin-binding activity. Proctolin-binding proteins were thus purified 1800-fold using standard chromatography.
Mazzocco
C
C
Laboratoire de Neuroendocrinologie de l'Insecte, Université Bordeaux I, Talence, France.
Puiroux
J
J
D000818:Animals; D001665:Binding Sites; D003058:Cockroaches / Q000737:chemistry*; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D019476:Insect Proteins / Q000737:chemistry / Q000302:isolation & purification*; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification; D009479:Neuropeptides / Q000737:chemistry; D009842:Oligopeptides / Q000737:chemistry*; D011485:Protein Binding; D012995:Solubility
D003902:Detergents; D019476:Insect Proteins; D008565:Membrane Proteins; D009479:Neuropeptides; D009842:Oligopeptides; C011536:proctolin