Tae Mook Cho

Stereospecific binding of narcotics to brain cerebrosides

Abstract Cerebrosides were shown to bind etorphine and naloxone stereo-specifically with high affinity. The relative potency of several narcotic analgesics in preventing the binding of etorphine and naloxone to cerebrosides correlated well with their reported intraventricular analgetic activity. The data indicate similarities between cerebroside sulfate and a purified opiate receptor from mouse brain which has been reported to be a proteolipid. Explanations for the apparent proteo-like behavior of the opiate receptor are provided.

Opiate binding to cerebroside sulfate: a model system for opiate-receptor interaction.

Abstract Cerebroside sulfate was shown to bind etorphine and levorphanol with high affinity. The relative potency of narcotic analgesics in preventing the binding of levorphanol to cerebroside sulfate correlated well with their reported analgetic activity. The data indicate similarities between cerebroside sulfate and a purified opiate receptor from mouse brain which has been reported to be a proteolipid. Some preliminary animal data also imply the involvement of CS in opiate action We, therefore, propose that CS may serve as a useful “receptor” model for the study of opiate-receptor interaction in vitro .

A model system for opiate-receptor interactions: Mechanism of opiate-cerebroside sulfate interaction

Abstract Narcotic analgetics were shown to bind cerebroside sulfate (CS) with high affinity. The binding correlated well with their pharmacological potency. In order to understand opiate receptor interaction at the molecular level, we have proposed the use of CS as a model opiate receptor. In these studies, our data indicate that the binding of opiates is determined by the heptane solubility of the drugs and their affinity to CS. The affinity of the agonist to CS is higher than that of its corresponding antagonist. The difference in affinity between an agonist and its corresponding antagonist is mainly due to the strength of electrostatic bond formed between the protonated nitrogen of the drug and the sulfate group of CS. Furthermore, we have concluded that narcotic agonist-CS complexes are more hydrophobic (intimate ion pairs formation) while the antagonist-CS complexes are more hydrophilic (hydrated ion pairs) in nature.

Solubilization of membrane bound opiate receptor from rat brain

Abstract Sonication of rat brain membranes for 9 minutes solubilized 35% of their stereospecific opiate binding activity; a second 9 minute sonication of the insoluble residue released an additional 21% of the original binding. The opiate binding properties of the solubilized material were highly similar to those of membrane bound receptor by a number of criteria, including affinity, effect of sodium, and the IC 50 of unlabeled opiates in displacing 3 H-etorphine binding. Moreover, storage of the solubilized receptor fraction for two weeks at −20°C did not significantly change the receptor binding. Sonication thus appears to be a useful first step in purifying the opiate receptor.

H3-cerebroside sulfate re-distribution induced by cation, opiate or phosphatidyl serine

Abstract Transfer of H 3 -cerebroside sulfate (CS) from aqueous phase to nonaqueous phases (heptane interface) was studied in the absence and presence of opiates, cations and phosphatidylserine. The degree of H 3 -CS re-distribution was dependent on the concentration of these substances used. The concentration of an opiate agonist (GPA-1657) required to increase H 3 -CS by 50% in the nonaqueous phase was much lower than that of its corresponding antagonist (GPA-2163) and the value for calcium was 100 times less than sodium. Opiate antagonist (GPA-2163) and phosphatidylserine inhibited the agonist induced re-distribution of H 3 -CS. Thus, the data seem to indicate that the distribution of H 3 -CS between these two phases was determined by hydrophobic-hydropholic balance of H 3 -CS and this balance was dependent on the counter ion pairing with CS. This finding is consistent with our previous observation that opiate agonist-CS complex was more hydrophobic than free CS of the CS-complex formed with opiate antagonist.

Isolation and purification of morphine receptor by affinity chromatography

Brain membranes were solubilized by sonication and Triton X-100 extraction and applied to an affinity column consisting of a 6-succinyl morphine derivative of Affi Gel-102. A fraction exhibiting high opiate binding was eluted by tris-buffer containing naloxone, CHAPS and NaCl. This fraction consisted of both proteins and acidic lipids. The opiate binding properties of this purified material exhibited many properties similar to those of membrane bound receptors of the u-type, including high affinity, stereospecificity, Na-effect and rank order in affinity for opiates. This opiate binding material was highly sensitive to both trypsin and N-ethylmaleimide. Based on the protein content of the isolated membrane receptor, a 3200-fold purification over the original brain P2 fraction was achieved.

Binding of dimethyltubocurarine to triphosphoinositide

DE ROBERTIS and his associates have described the binding of various cholinergic ligands to proteolipids isolated from biological materials. One of the studies was concerned with the binding of DMTC to one of the proteolipids isolated from the cat brain (DE ROBERTIS et al., 1969; DE ROBERTIS, 1974, 1975). In their experiments the DMTC-proteolipid complex was eluted with chloroform from a Sephadex LH-20 column. Acidic lipids, mainly PS, PI and CS, and also a little of TPI, DPI, have been known to be associated with proteolipid (FOLCH-PI, 1972). It was, therefore, our purpose to investigate the role of acidic lipids in the [14C]DMTC binding, and also to study the effect of deproteinization on the binding properties of the proteolipid.

The binding of 5-hydroxytryptamine to acidic lipids in isobutanol

Abstract— In order to examine the possibility that acidic lipids can account for the binding of 5‐hydroxy [3H]tryptamine (5‐HT) to brain tissue, the binding to six acidic lipids was studied using an isobutanol‐water partition method. With the exception of the polyphosphoinositides, all the acidic lipids examined bind saturably and with high affinity. The apparent dissociation constants of 5‐HT to the acidic lipids were as follows: phosphatidylserine, 0.4 μM; phosphatidic acid, 0.6μM; diphosphoinositide, 0.8 μM; cerebroside sulfate, 1.4 μM; monophosphoinositide, 1.9 μM; and triphosphoinositide, 10 μM. The high affinity of these lipids to 5‐HT raises the possibility of some role for them in serotonergic activity.

Effects of 5,5′-Dithiobis-(2-Nitrobenzoic Acid) on Opiate Binding to Both the Membrane-Bound Receptor and the Partially Purified Opiate Receptor

Abstract: Pretreatment of partially purified opiate receptor from rat brains with 5,5′‐dithiobis‐(2‐nitrobenzoic acid) (DTNB) decreased opiate agonist binding more effectively than that of antagonist. This agent, at a concentration that inhibits only 3H‐agonist binding, increases the IC50 values of agonists but not those of antagonists. We also observed similar effects of DTNB on opiate binding to the membrane‐bound receptor that are in good agreement with the published data. Moreover, there was an excellent correlation between the IC50 values of the two different preparations. However, opiate binding to the partially purified receptor was about a thousandfold more sensitive to DTNB than binding to this membrane‐bound receptor. Dithiothreitol, a sulfide bond reducing agent, reversed the effects of DTNB on the opiate binding.

EFFECTS OF MONOVALENT CATIONS ON CEREBROSIDE SULFATE BINDING TO OPIATE AGONISTS AND ANTAGONISTS

Monovalent cations, Li + , Na + , K + and Rb + , differentially affected the binding of the opiate agonists and antagonists to mixed micelles of cerebroside sulfate (CS) and nonacidic lipids. The cations were more effective in inhibiting the binding of agonists than that of antagonists. At a low concentration of Na + and Li + that inhibited 3 H-dihydromorphine binding, the 3 H-naloxone binding was enhanced specifically by Li + and Na + . Studies of the Na + effect on the binding revealed that the cation decreased more effectively the apparent affinity of agonist than that of antagonist. This effect was similar to the effect of Na+ on the brain tissue binding to opiate agonists and antagonists.