Jun 9, 1983 - lin-Arg6-Phe7 was found to be 3.5 to 1, whichis similar to that ob- served in mammalian proenkephalin-containing tissues. Together these data ...
Proc. Natl. Acad. Sci. USA Vol. 80, pp. 5772-5775, September 1983 Neurobiology
Evidence for a proenkephalin-like precursor in amphibian brain (opioid peptide/prohormone/protein evolution/HPLC)
DANIEL L. KILPATRICK, RICHARD D. HOWELLS, HANS-WERNER LAHM, AND SIDNEY UDENFRIEND Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110
Contributed by Sidney Udenfriend, June 9, 1983
ABSTRACT The mammalian proenkephalin-derived peptides [Leu]enkephalin, [Metlenkephalin, and [Met]enkephalin-Arg6Phe7 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-Gly7Leu8, 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.
tivities of [Met]enkephalin and [Leu]enkephalin (5), [Metlenkephalin-Arg6-Gly7-Leu8 (24), and [Met]enkephalin-Arg6-Phe7 with or without prior digestion with trypsin and carboxypeptidase B (25). The only peptide that we have found to crossreact significantly with the antiserum to [Met]enkephalin-Arg6-Phe7 is adrenal peptide B (>50%) which contains the heptapeptide sequence at its carboxyl terminus (26).
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 proenkephalin (1-6, 28), was not observed in toad brain extracts either by [Met]enkephalin radioimmunoassay after proteolytic digestion or by a specific and sensitive
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 (7-20). 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 we report the identification and characterization
octapeptide radioimmunoassay. To confirm the identities of the three opioid peptides that were observed, each was analyzed by HPLC on a phenyl reversed-phase column (Fig. 3). This support has a peptide selectivity that differs from that of the C18 support (compare calibration standards in Figs. 2 and 3). In each caser co-elution of immunoreactivity with the appropriate opioid peptide standard was observed. The identification of [Met]enkephalin-Arg6-Phe7 was further confirmed by sequential proteolytic digestion with trypsin and carboxypeptidase B (Fig. 4). Treatment with trypsin quantitatively converted it to [Met]enkephalin-Arg6 (Fig. 4A); treatment of the latter product with carboxypeptidase B yielded free [Met]enkephalin (Fig. 4B). These data are consistent with the structure of [Met]enkephalin-Arg6-Phe7. Previous studies in lower vertebrates have identified enkephalin immunoreactivities or, opiate receptor binding activities in crude tissue extracts without further attempts to determine their exact chemical nature. Measurements in crude extracts using radioimmunoassay or radioreceptor assay can be subject to nonspecific interference. Even after purification the observed activities may have represented peptides related to but structurally different from the enkephalins. Short of actual sequence analysis, we characterized both free [Metlenkephalin
of the mammalian proenkephalin-derived peptide [Met]enkephalin-Arg6-Phe7 together with [Met]enkephalin and [Leu]enkephalin in the brain of Bufo marinus& This provides direct evidence for the existence in a nonmammalian vertebrate of a proenkephalin that is structurally related to the mammalian prohormone.
METHODS Brains from 30 adult Bufo marinus (200-400 g) (West Jersey Biological, Wenonah, NJ) were removed immediately after decapitation and frozen in liquid nitrogen. Tissue extraction and Sephadex G-75 chromatography were carried out as described (21). Reversed-phase HPLC was performed on Nucleosil C18 (5-gm resin) and Nucleosil phenyl (7-ram resin) columns (4.6 X 250 mm) (Rainen Instruments, Ridgefield, NJ) with a fluorescamine monitoring system (22). Column fractions were assayed for opiate receptor binding activity (23) and for immunoreacThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisenent" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 5772
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Proc. Natl. Acad. Sci. USA 80 (1983)
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and free [Leu]enkephalin from amphibian brain with respect to size and mobility in two separate reversed-phase HPLC systems and further showed that their. quantitation by both opiate receptor binding assay and radioimmunoassay are identical. The identification of the heptapeptide [Met]enkephalin-Arg6-Phe7
FIG. 1. Sephadex G-75 chromatography of enkephalin-containing peptides from brain of Bufo marinus. [l, [Met]Enkephalin; !2, [L-eu]enkephalin. Tissues (6.7 g) were extracted with 1 M acetic acid/20 mM HCl containing 0.1% 2-mercaptoethanol and 1 ,4g each of phenylmethanesul-fonyl fluoride and pepstatin A per ml and chromatographed. Aliquots of column
was further confirmed by using a highly specific heptapeptide .radioimmunoassay and by sequential proteolytic conversion to [Met]enkephalin-Arg6 and free [Met]enkephalin. Mammalian proenkephalin contains four unique enkepha-
-lin sequences, -[Met]enkephalin, [Met]enkephalin-Arg6-Phe7,
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FIG. 2. Nucleosil C18 HPLC of toad brain low molecular weight enkephalincontaining peptides. The salt volume fractions from the Sephadex G-75 column were pooled and pumped directly onto the reversed-phase column. A gradient of 1-propanol (---) in 0.5 M pyridine acetate (pH 4.0) was used for peptide elution at a flow rate of 24 ml/hr. Aliquots from 1-ml fractions were assayed for [Met]enkephalin (0) and [Leu]enkephalin (ES) after digestion with trypsin and carboxypeptidase B; [Metlenkephalin-Arg6-Phe7 was assayed without prior enzymatic digestion by using a specific heptapeptide radioimmunoassay (0). Heavy arrows indicate the positions of 125I-labeled enkephalin internal calibration standards: (left to right) oxidized 125I-labeled [Met]enkephalin, reduced 1251I-labeled [Metlenkephalin, and 125I-labeled [Leu]enkephalin. Enk, enkephalin.
5774
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Proc. Natl. Acad. Sci. USA 80 (1983)
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