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ALTERATIONS IN THE BINDING OF [ 3 H]LEUCINE‐ENKEPHALIN TO STRIATAL MEMBRANES BY ENDOGENOUS FACTORS
Author(s) -
Rothman Richard B.,
Westfall Thomas C.
Publication year - 1979
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.1471-4159.1979.tb11721.x
Subject(s) - membrane , chemistry , centrifugation , morphine , trypsin , bovine serum albumin , ligand (biochemistry) , enkephalin , binding site , chromatography , levorphanol , biochemistry , biophysics , (+) naloxone , enzyme , biology , receptor , opioid , pharmacology
—Saturation binding studies with [ 3 H]leu‐enk ([tyrosyl‐3, 5‐ 3 H(N)] 5 leu‐enkephalin) revealed the presence of high and low affinity binding sites in a paniculate fraction derived from rat striatum. The binding of [ 3 H]leu‐enk to the high affinity component ( K D = 2.0 ± 0.3 nM) was sensitive to morphine and levorphanol, while the binding to the low affinity component ( K D = 21 ± 2 nM) was not. Incubation of the membranes, prior to assay for 30 min at 37°C, followed by centrifugation at 27, 000 g for 20 min in order to pellet the membranes allowed the detection of a factor, present in the high speed supernatant, which caused a dose‐dependent inhibition of the binding of [ 3 H]leu‐enk to the morphine‐sensitive and insensitive binding components. Investigations into the nature of the morphine‐insensitive binding component demonstrated that it was an artifact since it was not detectable when bound and free ligand were separated by centrifugation. Furthermore, [ 3 H]leu‐enk bound to Whatman glass fiber filters, used to collect bound ligand, in a morphine‐insensitive manner, and under conditions where the binding of [ 3 H]leu‐enk to the morphine sensitive component diluted proportionally with serial dilutions of the membranes, the binding to the morphine‐insensitive component did not. The factor present in the high speed supernatant did not dialyze and its effects were mimicked by either trypsin or soybean trypsin inhibitor, but not by bovine serum albumin. The apparent inhibition of the binding of [ 3 H]leu‐enk to these binding components is probably not of biological significance, but the fact that the artifactual morphine insensitive binding component of striatal membranes has been shown to decrease by 20–30% following lesions of the substantia nigra suggests that the influence of this endogenous factor must be controlled for.