Isolation of the opioid heptapeptide Met-enkephalin [Arg6,Phe7] from bovine adrenal medullary granules and striatum.

Abstract

Bovine adrenal chromaffin granules have been shown to contain, in addition to Met-enkephalin and Leu-enkephalin, at least three small peptides with opiate receptor activity. One of these adrenal peptides has been purified to homogeneity and its sequence was shown to be Met-enkephalin- [Arge, Phe7]. This heptapeptide was also found in beef striatal extracts in amounts comparable to those of Leu-enkephalin.The opioid pentapeptides Met-enkephalin and Leu-enkephalin were originally isolated from whole brain (1). When the Metenkephalin sequence was found at the NH2 terminus of the pituitary hormone (3-endorphin, it was assumed that Metenkephalin is derived from f3-endorphin. However, this relationship has never been established. Indeed, there have been studies that suggest a biosynthetic pathway for the brain enkephalins that does not include f3-endorphin as an intermediate (2)(3)(4).Recently, immunologic procedures have revealed the presence of opioid peptides in adrenal medulla, localized in chromaffin cells (5-9). We have not only corroborated these findings but also have found that the adrenal medulla contains more enkephalin-like material than does the brain. Furthermore, the medulla is also rich in several proteins and large peptides that yield opioid activity after treatment with trypsin (10). None of the proteins is related to f3-endorphin or its pituitary precursors.In the course of these studies we undertook the chemical characterization of those peptides in the medulla that are in the molecular weight range of the enkephalins and that show direct opioid activity. We found not only Met-and Leu-enkephalin, as expected, but also, at least three other small peptides with opioid activity. One of these unknown peptides has been purified to homogeneity and its sequence was shown to be Metenkephalin [Arg6, Phe7]. This peptide is found not only in the adrenal gland but also in the striatum in amounts comparable to those of Leu-enkephalin. Details of the isolation and characterization of this opioid heptapeptide are presented here. MATERIALS AND METHODSBovine adrenal glands were obtained from a local slaughterhouse and stored on ice until used (1-2 hr). The medullas were dissected out, and chromaffin granules were prepared by the procedure of Smith and Winkler (11). The isolated chromaffin granules were lysed in a solution (1:10, wt/vol) containing 1 M acetic acid, 20 mM HCI, 1 /Ag each of phenylmethylsulfonyl fluoride and pepstatin per ml, and 0.1% 2-mercaptoethanol. Membranes and cell debris were removed by centrifugation at 100,000 X g for 30 min, and proteins in the supernatant solution were precipitated by the addition of 50% (wt/vol) trichloroacetic acid to achieve a 10% final concentration. The precipitate was then removed by centrifugation at 26,000 X g for 15 min. After removal of trichloroacetic acid and lipids from the supernatant solution by ether extraction (three times with equal volumes), the sample was lyophilized. The residue was then dissolved in buffer and subjected to high-performance liq...

Release of enkephalins and enkephalin-containing polypeptides from perfused beef adrenal glands.

“…When the peak corresponding to enkephalins was subjected to high-performance liquid chromatography, it was found to contain [Met]enkephalinArg6,Phe7 in addition to the two enkephalins. This heptapeptide has also been found in extracts of brain (13).…”

Isolation and identification of enkephalins in pedal ganglia of Mytilus edulis (Mollusca).

“…Through additional results on biological assays and amino acid sequence analyses, the present report provides conclusive evidence demonstrating that [Met] MATERIALS AND METHODS Acid Extraction. The acid extraction of low molecular weight peptides from mussel pedal ganglia was carried out as described (7). The ganglia were homogenized with a Brinkmann Polytron homogenizer with four 3-sec bursts in an extraction solution (25 pedal ganglia per ml) containing 1 M acetic acid, 20 mM HCl, 1 ,ug each of phenylmethyl-sulfonyl fluoride and pepstatin A per ml, and 1% 2-mercaptoethanol.…”

Distribution, Isolation and Sequence Analysis of the C‐Terminal Heptapeptide of Pro‐Enkephalin A (YGGFMRF) from the Ovine Median Eminence

“…1 ) as shown by chemical isolation (4) or cDNA cloning from a number of species including man (5), cow (6, 7) and toad (8).…”