Evidence for a proenkephalin-like precursor in amphibian brain.

Abstract

The mammalian proenkephalin-derived peptides [Leu]enkephalin, [Metlenkephalin, and [Met]enkephalin-Arg6-Phe7 were identified in acid extracts of the brain of Bufo marinus by using reversed-phase HPLC and specific radioimmunoassays.[Met]Enkephalin was the predominant opioid peptide present (270 pmol/g). In contrast, the octapeptide [Met]enkephalin-Arg6-Gly7-Leu8, which is also derived from mammalian proenkephalin, was not detected. The ratio of free [Met]enkephalin to [Met]enkephalin-Arg6-Phe7 was found to be 3.5 to 1, which is similar to that observed in mammalian proenkephalin-containing tissues. Together these data (i) indicate that amphibian brain contains a proenkephalin related to the mammalian precursor and (ii) establish the existence of enkephalins and proenkephalin-derived enkephalincontaining peptides in a submammalian species.It is now well established that the opioid pentapeptides [Met]-enkephalin and [Leu]enkephalin are generated from a common precursor molecule, proenkephalin (1, 2). Evidence for this biosynthetic pathway has been found in all mammalian species examined (3-6). There also have been numerous reports of enkephalin-like substances in lower vertebrate and invertebrate tissues (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Although it has been assumed that these materials represent enkephalins, such studies were based mainly on immunohistochemistry or radioligand displacement assays performed on crude tissue extracts. In no case has there been a critical biochemical characterization of nonmammalian enkephalin-like material. To our knowledge, the presence of free enkephalins and the proenkephalin biosynthetic pathway in submammalian species has yet to be established.We have been examining the distribution of enkephalins and proenkephalin-derived peptides in several submammalian species by gel filtration and high-performance liquid chromatography. Here RESULTS AND DISCUSSION Sephadex G-75 chromatography of toad brain acid extracts revealed the presence of significant amounts of enkephalin immunoreactivity (Fig. 1). After digestion with trypsin and carboxypeptidase B, enkephalin activities appeared in both the low and high molecular weight regions.[Met]Enkephalin-immunoreactive peptides were clearly predominant. The enkephalin-containing peptides present in the Sephadex G-75 salt volume fractions were resolved by using reversed-phase HPLC on a C18 column. Several enkephalin-immunoreactive peptides were detected (Fig. 2). By far the most abundant peptide was eluted in the position of free [Met]enkephalin. Immunoreactive peaks corresponding to free [Leu]enkephalin and [Met]-enkephalinArg6-Phe7 were observed also. In mammals, the latter peptide is derived from the processing of proenkephalin (1-6, 27). All three enkephalin immunoreactive peaks also exhibited opiate receptor binding activities proportional to their immunoreactivities (Table 1). Interestingly, the octapeptide [Met]enkephalin-Arg6-Gly7-Leu8, which also is produced through the processing of mammalian proenkephal...

Comparative Neuroendocrinology of Gut Peptides

“…Evidently, in the lines giving rise to modern mammals and amphibians, there have been substitutions of Leu for Met in one of the enkephalin species and of Tyr for Leu in the octapeptide variant. These observations are based on gene sequences but are compatible with reports that Met-enkephalin Arg6Gly7Leus is absent from extracts of the brain of Bufo marinus (195). Extracts of Bufo brain do, however, contain Leu-enkephalin, which presumably arises from prodynorphin and is consistent with the observation of other prodynorphin-related peptides in amphibia (63).…”

Analyzing the evolution of the opioid/orphanin gene family

“…When these studies were begun in the 1980s, it was assumed that: (a) met-enkephalin and leu-enkephalin would be co-localized in neurons; (b) the molar ratio of metenkephalin to leu-enkephalin in CNS extracts would be approximately 4:1; and (c) both of these opioids would be encoded by the same precursor. Steady-state analyses of non-mammalian brain extracts (Kilpatrick et al, 1983;Lindberg & White, 1986), and immunocytochemical studies (Vallarino et al, 1994(Vallarino et al, , 1998 indicated that the evolution of the enkephalinergic system was more complicated than had been anticipated.…”

Identification of a Fourth Opioid Core Sequence in a Prodynorphin cDNA Cloned from the Brain of the Amphibian, <i>Bufo marinus</i>: Deciphering the Evolution of Prodynorphin and Proenkephalin

“…In an earlier study, higher than expected levels of met-enkephalin were detected in the brain of B. marinus [17]relative to leu-enkephalin. Subsequent studies have shown that anuran amphibian proenkephalin lacks a leu-enkephalin sequence [18, 19, 20, 21], hence it was assumed that the met-enkephalin present in the brains of anuran amphibians came exclusively from proenkephalin.…”