A variety of enzymatic treatments have been examined with regard to their effect on stereospecific [3H]naloxone binding to rat brain homogenates. Opiate receptor binding is sensitive to very low concentrations of trypsin (EC 18.104.22.168) and chymotrypsin (EC 22.214.171.124). Trypsin appears to decrease the number of opiate receptor binding sites, while chymotrypsin primarily lowers their affinity for opiates. Receptor binding is drastically reduced by very low concentrations of phospholipase A (EC 126.96.36.199), is decreased by higher concentrations of phospholipase C (EC 188.8.131.52), and appears relatively insensitive to phospholipase D (EC 184.108.40.206) and neuraminidase (EC 220.127.116.11). Small amounts of RNase (EC 18.104.22.168) and DNase (EC 22.214.171.124) are without effect. Trypsin and chymotrypsin decrease receptor binding in a biphasic fashion, suggesting the presence of more than one population of sites sensitive to proteolysis. By contrast, the phospholipases degrade binding in a monophasic fashion. Thus the opiate receptor appears to be a membrane-bound complex whose stereospecific binding is dependent upon the integrity of both proteins and phospholipids.
ACKNOWLEDGMENTS The authors gratefully thank Drs. Pedro Cuatrecasas, Donald Coffey, Indu Parikh, and Steven March for their helpful discussions and suggestions, and Dr. Alan Goldberg for measuring choline levels.
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