BACKGROUND: Purification of opioid receptor is mandatory to improve opiate analgesic medication. Recently, it was reported that sodium ion increased the number of opioid binding sites for opioid antagonist. The importance of sodium ions lead us to design appropriate affinity chromatography and binding assay for the successful purification of mu-opioid receptor to homogeneity. METHODS: Opioid receptor was solubilized from rat brain membranes with a mixture of the detergents, CHAPS and digitonin, in the presence of protease inhibitors and 1M NaCl. The solubilized material was passed through an opioid antagonist(10cd) affinity column and a wheat germ agglutinin(WGA) column, set up in series, to obtain a partially purified receptor preparation. The partially purified receptor was further purified by repeating the affinity and lectin chromatography with smaller size column. RESULTS: Binding of opioid antagonist [H]diprenorphine to the partially purified or purified receptors was dependent upon the presence of sodium ions. The purified receptor showed diffuse band with a medium molecular mass of 62KD upon electrophoresis. The average specific binding activity of the purified receptor was 18.8+/-2.3 pmol/mcg protein. CONCLUSIONS: Opioid agonists and antagonists either do not bind or bind with low affinity to G protein-dissociated free opioid receptors in the absence of sodium ions. However, the free opioid receptors have a high affinity for antagonists but not agonists in the presence of sodium ions.
Animals ; Binding Sites ; Brain ; Chromatography ; Chromatography, Affinity ; Detergents ; Digitonin ; Electrophoresis ; Ions ; Membranes ; Protease Inhibitors ; Rats ; Receptors, Opioid* ; Sodium* ; Triticum

Mammalian acetyl-CoA carboxylase (ACC) is present in two isoforms, alpha and beta, both of which catalyze formation of malonyl-CoA by fixing CO2 into acetyl-CoA. ACC-alpha is highly expressed in lipogenic tissues whereas ACC-beta is a predominant form in heart and skeletal muscle tissues. Even though the tissue-specific expression pattern of two ACC isoforms suggests that each form may have a distinct function, existence of two isoforms catalyzing the identical reaction in a same cell has been a puzzling question. As a first step to answer this question and to identify the possible role of ACC isoforms in myogenic differentiation, we have investigated in the present study whether the expression and the subcellular distribution of ACC isoforms in H9c2 cardiac myocyte change so that malonyl-CoA produced by each form may modulate fatty acid oxidation. We have observed that the expression levels of both ACC forms were correlated to the extent of myogenic differentiation and that they were present not only in cytoplasm but also in other subcellular compartment. Among the various tested compounds, short-term treatment of H9c2 myotubes with insulin or okadaic acid rapidly increased the cytosolic content of both ACC isoforms up to 2 folds without affecting the total cellular ACC content. Taken together, these observations suggest that both ACC isoforms may play a pivotal role in muscle differentiation and that they may translocate between cytoplasm and other subcellular compartment to achieve its specific goal under the various physiological conditions.
Acetyl-CoA Carboxylase/metabolism* ; Acetyl-CoA Carboxylase/drug effects ; Animal ; Cell Differentiation/drug effects ; Cell Line ; Cell Membrane Permeability ; Chromones/pharmacology ; Cytosol/enzymology* ; Cytosol/drug effects ; Digitonin/pharmacology ; Immunoblotting ; Insulin/pharmacology* ; Isoenzymes ; Morpholines/pharmacology ; Myocardium/cytology ; Okadaic Acid/pharmacology* ; Phosphorylation ; Rats