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Analysis of peptide biosynthesis in the neurointermediate lobe of Xenopus laevis using high-performance liquid chromatography: occurrence of small bioactive products
Comp. Biochem. Physiol. Vol. 67B, pp. 493 to 497
0305-0491/80"1201-0493502.00/0
© Pergamon Press Ltd 1980. Printed in Great Britain
ANALYSIS OF PEPTIDE BIOSYNTHESIS IN THE NEUROINTERMEDIATE LOBE OF X E N O P U S L A E V I S USING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY: OCCURRENCE OF SMALL BIOACTIVE PRODUCTS GERARD J. M. MARTENS, B. G. JENKS and A. P. VAN OVERBEEKE Department of Zoology, Faculty of Science, Catholic University, Toernooiveld, 6525 ED Nijmegen, The Netherlands (Received 24 March 1980) Abstrae~l. Peptide biosynthesis in neurointermediate lobes of black adapted Xenopus laevis was studied using pulse-chase incubation and reversed-phase, high-performance liquid chromatographic analysis. 2. During the pulse period one major product was synthesized, which was subsequently converted to 12 chase peptides, suggesting a precursor-product mode of biosynthesis for this tissue. 3. Chase peptides I, II and IV possessed high melanotropic activity. Alpha-MSH did not appear to be among the chase peptides. Peptide II had also high corticotropic activity. 4. Peptides I and II are probably small, since they were TCA-soluble and ran faster on acid-urea gels than ~-MSH. They may, however, well be structurally related to this latter hormone.
INTRODUCTION Many amphibians, when placed on a black background, darken as a result of the dispersion of the black pigment melanin in the dermal melanophores. This dispersion is controlled by a melanophore stimulating hormone (MSH) from the pars intermedia of the pituitary gland. Electrophoretic analysis of amphibian pituitary glands has revealed the presence of a number of melanotropic peptides (Burgers, 1961; Preslock & Brinkley, 1970a, b; Hopkins, 1972; Loh & Gainer, 1977). Pulse-chase analysis of biosynthetic events in neurointermediate lobes of the African clawed toad, Xenopus laevis (Loh, 1979; Jenks & van Overbeeke, 1980), suggests that most, if not all, of the melanotropic peptides are derived from a precursor that is a prohormone to corticotropin (ACTH) and endorphins. While it has been generally accepted that ~t-MSH represents the melanotropic peptide in amphibians, Jenks & van Overbeeke (1980) obtained results pointing towards the possibility that this product is not synthesized in fully black adapted Xenopus laevis. They suggest that one of the other chase peptides might act as the melanotropic factor responsible for maintaining the animal in the darkened state. Although in our laboratory important information on the complex pathways of peptide biosynthesis has been obtained by employing gel electrophoresis, we have indications that some of the neurointermediate lobe peptides were co-migrating on the gel, thus indicating the need for improved separation methods, preferably with higher resolution. Nice & O'Hare (1979), attempting to separate synthetic corticotropinand endorphin-like peptides, successfully employed reversed-phase high-performance liquid chromatography (HPLC). We wish to report on the application of HPLC to 493 c.B.P. 67/4B
A
analyse peptide biosynthesis in neurointermediate lobes of black adapted Xenopus laevis. In vitro pulse and pulse-chase experiments were performed. The melanotropic and corticotropic activity of the various peptides separated with HPLC were analysed with appropriate bioassays. MATERIALS AND METHODS
Animals Adult male Xenopus laevis were bred and reared in the Department of Zoology, University of Nijmegen. In all experiments neurointermediate lobes from fully black adapted animals were used. The conditions for adaptation to a black background were the same as previously described (Jenks & van Overbeeke, 1980). American lizards, Anolis earolinensis, to be used for the MSH bioassay, were purchased from Carolina Biological Supply Company (Burlington, North Carolina, U.S.A.). Adult male Wistar rats, to be used for the preparation of adrenal cell suspensions in the ACTH bioassay, were obtained from the Central Animal Laboratory, University of Nijmegen. Pulse and pulse-chase incubations Neurointermediate lobes of black adapted Xenopus were preincubated for l hr in 500/~1 incubation medium ( l l 2 m M NaCI, 2mM KCI, 2raM CaC12, 15mM Hepes, Bovine serum albumin (BSA) 0.3 mg/ml, glucose 2 mg/ml, pH 7.38). The 30-min pulse incubation was done in the presence of 40/tCi [3H]lysine (New England Nuclear, sp. act. 60 Ci/mmol) on a Dubnoff metabolic shaker (22°C). The lobes were then homogenized in 500 #1 0.1 M acetic acid (HAc) in an all-glass homogenizer, the homogenate was centrifuged (5 min, 10,000 g, Beckman microfuge) and the supernatant was analysed with HPLC. In pulse-chase incubations, after the 30-min pulse in [3H]lysine, the lobes were chase-incubated for 6 hr in medium containing 5 mM L-lysine (Calbiochem, San Diego, California, U.S.A.). Following the chase incubation, the lobes were homogenized,
494
GERARD J. M. MARTENS, B. G. JENKS and A. P. VAN OVERBEEKE
the homogenate was centrifuged and the supernatant was injected onto the HPLC column. To determine which of the newly synthesized products contained the amino acid leucine, neurointermediate lobes were pulse-incubated for 6 hr in medium containing 40 pCi [3H]leucine (New England Nuclear, sp. act. 120 Ci/mmol) and lobe extracts, prepared as described above, were submitted to HPLC analysis.
High-performance liquid chromatography A Spectra Physics high-performance liquid chromatograph (model SP 8000) equipped with manual injector and ternary gradient system was used (Spectra Physics B.V., Eindhoven, The Netherlands). The stainless-steel column (250 × 4.6 mm i.d.) was packed with Spherisorb 10 ODS (Chrompack B.V., Middelburg, The Netherlands). The linear gradient was established with two solvents. Solvent A consisted of 0.5 M formic acid, 0.14 M pyridine (pH 3.0) and solvent B was l-propanol. All solvents were purified on a 0.45/am filter (type HA, Millipore B.V., Brussels, Belgium). Preliminary experiments were conducted concerning the gradient profile to optimize resolution of neurointermediate lobe peptides. The final gradient elution adopted, is included on the chromatograms. During the gradient elution the pressure increased from 700 to 2000 p.s.i. The polypeptides were concentrated on the head of the column during a 2-min loading phase. Equilibration of the column to the initial conditions lasted about 10 min. In all experiments, the sample volume brought on the column was 500/~1 of lobe extract. The column was eluted with a flow rate of 2 ml/min and 0.5-min fractions were collected with a fraction collector (LKB Redirac, model 2112). To each fraction 4 ml Aqua luma (Baker Chemicals B.V., Deventer, The Netherlands) was added and the samples were counted on a liquid scintillation analyser (Philips, model PW 4510). To determine the elution time of ct-MSH on the HPLCsystem, synthetic ct-MSH (Peninsula, San Carlos, California, USA) was dissolved in 250 #1 solvent A and detected in the eluate fractions at 280nm (Zeiss spectrophotometer, model PM 6).
Electrophoretic analysis To compare the results of the HPLC analysis with previous data of electrophoretic analysis, some of the labelled peptides separated by HPLC were submitted to electrophoresis. To the appropriate HPLC fractions, 50/~g BSA was added, the fractions were freeze-dried and then analysed by the acid-urea electrophoretic procedure described earlier (Jenks et al., 1979). It was evident that some of the peptides that were resolved by HPLC had not been found in the previous electrophoretic study (Jenks & van Overbeeke, 1980). As in this last investigation trichloroacetic acid (TCA) was used to prepare lobe homogenates, the possibility was considered that these new "HPLC peptides" constitute a TCAsoluble pool. A lobe extract from a pulse-chase incubation using [3H]lysine was divided into two equal aliquots. One aliquot was precipitated with 20?o TCA and the precipitate analysed with electrophoresis. The other aliquot was freeze-dried and the lyophilized extract was submitted to electrophoretic analysis.
RESULTS
H P L C analysis of pulse and pulse-chase incubations H P L C analysis of the incorporation of [3H]lysine into neurointermediate lobes of Xenopus laevis during the pulse period, showed one major labelled product (peak XI), eluting at 50 min (Fig. 1, filled circles). The peaks eluting in the first 10 min were [3H]lysine and contaminations in the [-3H]lysine preparation. After a 6-hr chase incubation, peak XI was greatly reduced and at least 12 other products (peaks I-X, XII and XIII) could be resolved on the reversed-phase column with the gradient system used (Fig. 1, open circles).
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MSH bioassay Neurointermediate lobes were homogenized in 500 #1 0.1 M HAC, the homogenate was centrifuged, and the supernatant was submitted to HPLC. To the 1-min HPLC fractions, 50 #g BSA was added and the samples were freeze-dried. The Anolis carolinensis skin bioassay, as described by Tilders et al. (1975), was used with minor modifications. Each freeze-dried fraction was dissolved in 50/A A. carolinensis Ringer's solution and, as a preliminary survey for melanotropic activity, 20/~1 of this solution was added to 100/A Ringer's solution containing a small piece of A. carolinensis skin. Those fractions which showed melanotropic activity (darkening of the skin) were further diluted to determine their relative potency, i.e. the dilution factor whereby the sample was capable of visibly changing the bright green colour of the skin sample into a greenishbrown within 15 rain.
ACTH bioassay HPLC-fractions, 0.5min, were prepared as described above under "MSH bioassay". ACTH bioactivity of individual fractions was determined using adrenal cell suspensions following the Sayers assay (Sayers et al., 1971) in a modification as reported by Goverde et al. (1980). The sensitivity of this modified bioassay was approx 1 pg per ml cell suspension, using human ACTH 1-39 (CIBA-Geigy Ltd, Basel, Switzerland) as a standard.
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Fig. 1. High-performance liquid radiochromatogram of neurointermediate lobe peptides of black adapted Xenopus laevis. The tissue was pulse incubated for 30 min (filled circles) or pulse-chase incubated (30 min pulse, 6-hr chase; open circles) with [3H]lysine. Separation at ambient temperature was on Spherisorb 10 ODS (250 x 4.6 mm i.d.) with a flow rate of 2 ml/min; fractions were collected every 30 sec. Primary solvent (A): 0.5 M formic acid4).14 M pyridine (pH 3.0). Secondary solvent (B): 1-propanol.
495
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Fig. 2(a) Melanotropic activity of a high-performance liquid chromatogram prepared from neurointermediate lobe extract of black adapted Xenopus laevis. Activity of 1-min fractions was determined (see also Materials and Methods). (b) Adrenocorticotropic activity of a highperformance liquid chromatogram prepared from neurointermediate lobe extract of black adapted Xenopus laevis. Activity of 0.5-min fractions was determined (see also Materials and Methods). The chromatographic conditions were the same as given in Fig. 1.
It could be established that, following the chase period, only a very small amount of product XI was present in the incubation medium (data not shown). Release of newly synthesized products is currently under investigation.
Melanotropic and corticotropic activity Using the Anolis carolinensis skin bioassay it was shown that there were three major peaks of melanotropic activity (Fig. 2a). The elution times for these products corresponded exactly with those of chase peptides I, II and IV. Synthetic ct-MSH eluted from the HPLC column at 17 rain, which is between peaks II and IV (data not shown). The bioassay for adrenocorticotropic activity demonstrated the presence of a number of minor peaks and one major peak (Fig. 2b). The elution time of the major peak corresponded exactly with that of chase peptide II. Acid-urea gel electrophoresis The electrophoretic pattern for the freeze-dried extract of 30-min pulse, 6-hr chase incubated neurointermediate lobes is illustrated in Fig. 3 (open circles). This pattern is essentially the same as that reported by Jenks & van Overbeeke (1980) with the exception that there are three new products, namely W, Y and Z, which migrate to gel slices 42, 45-46 and 48 respectively. Analysis of the TCA precipitated lobe extract showed these peaks W, Y and Z to be absent from the electrophoretogram (Fig. 3, filled circles), which indicates that products W, Y and Z are TCAsoluble. Electrophoresis-~f HPLC-peaks I, II and IV,
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Fig. 3. Acid-urea gel electrophoresis of products synthesized in neuro-intermediate lobes of black adapted Xenopus laevis during a 30-min pulse, 6-hr chase incubation using [3H]lysine. Part of the lobe extract was freezedried (open circles). The remainder was TCA-precipitated; the precipitate produced essentially the same electrophoretogram, with the exception of the area between gel slices 40 and 50 (filled circles).
which corresponded to peaks of major bioactivity (Fig. 2), revealed that these products co-migrated with Z, Y and E respectively. Product VI appeared to comigrate with the TCA-soluble peptide W. The major labelled product of the pulse incubation, product XI, corresponded with product A on the acid-urea gel.
[ 3H] Leucine incorporation Comparison of the peak pattern following [3H]leucine incorporation (Fig. 4) with that after [3H]lysine
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Fig. 4. High-performance liquid radiochromatogram of neurointermediate lobe peptides of black adapted Xenopus laevis. The tissue was pulse incubated for 6hr with [3H]leucine. Value for peak XI was 1662cpm. The chromatographic conditions were the same as given in Fig. 1. Insert: Same extract, but chromatographed with a less steep gradient (see also text).
496
GERARDJ. M. MARTENS,B. G. JENKSand A. P. VAN OVERBEEKE
incorporation (Fig. 1) strongly indicates that peaks I and II do not contain leucine. To improve the resolution of the area concerned, the same leucine-labelled sample was analysed using a less steep gradient (Fig. 4, insert). It was confirmed that products I and II, present after [3H]lysine incorporation, were absent from the chromatogram. DISCUSSION HPLC, in general, has some important advantages over electrophoretic techniques: (1) higher capacity for sample loading which no longer necessitates TCA precipitation or freeze-drying of the extract, (2) shorter separation time, (3) higher resolution and (4) possibility to adjust the resolution in selected areas. In using HPLC with a reversed-phase column, we did, however, experience one problem, namely that initially product XI did not elute from the column. In our opinion, this may have been due to a loading effect, such as irreversible binding to residual silanol groups. This suggestion was earlier proposed by O'Hare & Nice (1979) to explain the lack of elution of some high-molecular weight proteins from a reversedphase column. The fact that in our study after repeated analyses using the same column, peak XI did eventually elute, suggests that a conditioning of the column occurred, presumably through saturation of the silanol groups. The results of our HPLC analysis of pulse and pulse-chase incubations revealed the synthesis in the neurointermediate lobe of 13 products. Since peak XI represented the only major product during the pulse incubation and since this peak, furthermore, became reduced during the chase period, simultaneous with the appearance of the 12 other peaks, it may be concluded that product XI most likely functions as a precursor for the 12 chase peptides. This confirms the existence of a precursor-product mode of biosynthesis in the neurointermediate lobe, reported by Jenks & van Overbeeke (1980). It is therefore not surprising that product XI appeared to have the same electrophoretic properties as product A, considered to be the precursor by Jenks & van Overbeeke (1980). Three of the chase peptides, namely I, II and IV, had high melanotropic activity but, at least on the basis of elution time, none of these corresponded to c~-MSH. The lack of any newly synthesized peptide corresponding to c~-MSH seems to support the suggestion by Jenks & van Overbeeke (1980) that c~-MSH is not a major biosynthetic product in fully black adapted X e n o p u s laevis. The melanotropic peptides I and II are most likely the same products as peptides Z and Y, respectively, and they are, therefore, TCA-soluble. Moreover, on acid-urea gels these two peptides ran faster than c~-MSH (a-MSH migrates to gel slices 34-35, Jenks et al., 1979). Both their solubility in TCA and their electrophoretic behaviour point to the likelihood that peptides I and II are small peptides. The occurrence in X e n o p u s laevis of a TCAsoluble, melanotropic peptide has been reported earlier by Loh & Gainer (1977). The amino acid composition of the peptides I and II, discovered in our study, remains to be elucidated. The fact, however, that they have melanotropic activity indicates that they contain the "message sequence" ACTH 4 ~o
(Schwyzer & Eberle, 1977). Thus, they may well be structurally related to ~-MSH. The absence of the amino acid leucine in peptides I and II, too, is reminiscent of ~-MSH, since leucine is not found in any of the known ~-MSH structures. It is surprising that the major part of the corticotropic activity eluted with the small, TCA-soluble, melanotropic peptide II and not with any of the larger peptides. Schwyzer et al., (1971) demonstrated that small peptides, namely fragments of ACTH 1 24 possess some corticotropic activity, but far less than the complete molecule (ACTH 1 24). If, indeed, the corticotropic potency of peptide II were low, then peptide II must have been present in a high concentration. Alternatively, it might be a new, small peptide with a high corticotropic potency. The nature of the small bioactive peptides I and II is currently under investigation. Acknowledgements We thank Messrs H. J. M. Goverde and G. J. Pesman for performing the ACTH bioassays and Mrs E. M. Jansen-Hoorweg for expert typing.
REFERENCES
BUROERS A. (3. J. (196l) Occurrence of three electrophoretic components with melanocyte-stimulating activity in extracts of single pituitary glands from ungulates. Endocrinology 68, 698-703. GOVERDEH. J. M., PESMANG. J. & BENRAADTH. J. (1980) Improved sensitivity to adrenocorticotropin after purification and preincubation of rat adrenal cells. Acta Endocr. in press. HOPKINS C. R. (1972) The biosynthesis, intracellular transport, and packaging of melanocyte-stimulating peptides in the amphibian pars intermedia. J. Cell Biol. 53, 642-653. JENKS B. G., MEULEPASW. J. A. M., SOONSP. J. A. & VAN OVERBEEKE A. P. (1979) Biosynthesis of MSH and related peptides in the pars intermedia of the mouse: a pulse-chase analysis. Mol. cell. Endocr. 13, 149 158. JENKS B. G. & VAN OVERBEEKEA. P. (1980) Biosynthesis and release of neurointermediate lobe peptides in the aquatic toad, Xenopus laevis, adapted to a black background. Comp. Biochem. Physiol. 66, in press. Loll Y. P. & GAINER H. (1977) Biosynthesis, processing, and control of release of melanotropic peptides in the neurointermediate lobe of Xenopus laevis. J. gen. Physiol. 70, 37 58. LOll Y. P. (1979) Immunological evidence for two common precursors to corticotropins, endorphins, and melanotropins in the neurointermediate lobe of the toad pituitary. Proc. natn. Acad. Sci. U.S.A. 76, 796-800. NICE E. C. & O'HARE M. J. (1979) Simultaneous separation of ,8-1ipotrophin, adrenocorticotropic hormone, endorphins and enkephalins by high-performance liquid chromatography. J. Chromat. 162, 401-407. O'HARE M. J. & NICE E. C. (1979) Hydrophobic highperformance liquid chromatography of hormonal polypeptides and proteins on alkylsilane-bonded silica. J. Chromat. 171, 209 226. PRESLOCKJ. P. & BRINKLEYH. J. (1970a: Chemical quantification of melanophore stimulating substances using polyacrylamide gel disc-electrophoresis. Life Sci. 9, 215 -228. PRESLOCK J. P. & BRINKLEYH. J. (1970b) Melanophore and adrenocortical stimulating activities of substances from the pars intermedia, pars distalis, hypothalamus and cerebral cortex of the frog, Rana pipiens. Limb Sci. 9, 1369-1380.
Peptide synthesis in neurointermediate lobe of Xenopus laevis SAYERS G., SWALLOW R. L. & GIORDANO N. D. (1971) An improved technique for the preparation of isolated rat adrenal cells. A sensitive, accurate and specific method for the assay of ACTH. Endocrinology 88, 1063-1068. SCHWYZER R., SCHILLER P., SEELIG S. t~ SAYERSG. (1971) Isolated adrenal cells: log dose response curves for steroidogenesis induced by ACTH, 24, ACTH,_,o, ACTH4_lo, and ACTH 5 lo. FEBS Lett. 19, 229-231.
497
SCHWYZER R. & EBERLEA. (1977) On the molecular mechanism of ct-MSH receptor interactions. Front. Hormone Res. 4, 18-25. TILDERS F. J. H., VAN DELFT A. M. L. & SMELIK P. G. (1975) Re-introduction and evaluation of an accurate high capacity bioassay for melanocyte stimulating hormone using the skin of Anolis carolinensis in vitro. J. Endocr. 66, 165 175.
0305-0491/80"1201-0493502.00/0
© Pergamon Press Ltd 1980. Printed in Great Britain
ANALYSIS OF PEPTIDE BIOSYNTHESIS IN THE NEUROINTERMEDIATE LOBE OF X E N O P U S L A E V I S USING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY: OCCURRENCE OF SMALL BIOACTIVE PRODUCTS GERARD J. M. MARTENS, B. G. JENKS and A. P. VAN OVERBEEKE Department of Zoology, Faculty of Science, Catholic University, Toernooiveld, 6525 ED Nijmegen, The Netherlands (Received 24 March 1980) Abstrae~l. Peptide biosynthesis in neurointermediate lobes of black adapted Xenopus laevis was studied using pulse-chase incubation and reversed-phase, high-performance liquid chromatographic analysis. 2. During the pulse period one major product was synthesized, which was subsequently converted to 12 chase peptides, suggesting a precursor-product mode of biosynthesis for this tissue. 3. Chase peptides I, II and IV possessed high melanotropic activity. Alpha-MSH did not appear to be among the chase peptides. Peptide II had also high corticotropic activity. 4. Peptides I and II are probably small, since they were TCA-soluble and ran faster on acid-urea gels than ~-MSH. They may, however, well be structurally related to this latter hormone.
INTRODUCTION Many amphibians, when placed on a black background, darken as a result of the dispersion of the black pigment melanin in the dermal melanophores. This dispersion is controlled by a melanophore stimulating hormone (MSH) from the pars intermedia of the pituitary gland. Electrophoretic analysis of amphibian pituitary glands has revealed the presence of a number of melanotropic peptides (Burgers, 1961; Preslock & Brinkley, 1970a, b; Hopkins, 1972; Loh & Gainer, 1977). Pulse-chase analysis of biosynthetic events in neurointermediate lobes of the African clawed toad, Xenopus laevis (Loh, 1979; Jenks & van Overbeeke, 1980), suggests that most, if not all, of the melanotropic peptides are derived from a precursor that is a prohormone to corticotropin (ACTH) and endorphins. While it has been generally accepted that ~t-MSH represents the melanotropic peptide in amphibians, Jenks & van Overbeeke (1980) obtained results pointing towards the possibility that this product is not synthesized in fully black adapted Xenopus laevis. They suggest that one of the other chase peptides might act as the melanotropic factor responsible for maintaining the animal in the darkened state. Although in our laboratory important information on the complex pathways of peptide biosynthesis has been obtained by employing gel electrophoresis, we have indications that some of the neurointermediate lobe peptides were co-migrating on the gel, thus indicating the need for improved separation methods, preferably with higher resolution. Nice & O'Hare (1979), attempting to separate synthetic corticotropinand endorphin-like peptides, successfully employed reversed-phase high-performance liquid chromatography (HPLC). We wish to report on the application of HPLC to 493 c.B.P. 67/4B
A
analyse peptide biosynthesis in neurointermediate lobes of black adapted Xenopus laevis. In vitro pulse and pulse-chase experiments were performed. The melanotropic and corticotropic activity of the various peptides separated with HPLC were analysed with appropriate bioassays. MATERIALS AND METHODS
Animals Adult male Xenopus laevis were bred and reared in the Department of Zoology, University of Nijmegen. In all experiments neurointermediate lobes from fully black adapted animals were used. The conditions for adaptation to a black background were the same as previously described (Jenks & van Overbeeke, 1980). American lizards, Anolis earolinensis, to be used for the MSH bioassay, were purchased from Carolina Biological Supply Company (Burlington, North Carolina, U.S.A.). Adult male Wistar rats, to be used for the preparation of adrenal cell suspensions in the ACTH bioassay, were obtained from the Central Animal Laboratory, University of Nijmegen. Pulse and pulse-chase incubations Neurointermediate lobes of black adapted Xenopus were preincubated for l hr in 500/~1 incubation medium ( l l 2 m M NaCI, 2mM KCI, 2raM CaC12, 15mM Hepes, Bovine serum albumin (BSA) 0.3 mg/ml, glucose 2 mg/ml, pH 7.38). The 30-min pulse incubation was done in the presence of 40/tCi [3H]lysine (New England Nuclear, sp. act. 60 Ci/mmol) on a Dubnoff metabolic shaker (22°C). The lobes were then homogenized in 500 #1 0.1 M acetic acid (HAc) in an all-glass homogenizer, the homogenate was centrifuged (5 min, 10,000 g, Beckman microfuge) and the supernatant was analysed with HPLC. In pulse-chase incubations, after the 30-min pulse in [3H]lysine, the lobes were chase-incubated for 6 hr in medium containing 5 mM L-lysine (Calbiochem, San Diego, California, U.S.A.). Following the chase incubation, the lobes were homogenized,
494
GERARD J. M. MARTENS, B. G. JENKS and A. P. VAN OVERBEEKE
the homogenate was centrifuged and the supernatant was injected onto the HPLC column. To determine which of the newly synthesized products contained the amino acid leucine, neurointermediate lobes were pulse-incubated for 6 hr in medium containing 40 pCi [3H]leucine (New England Nuclear, sp. act. 120 Ci/mmol) and lobe extracts, prepared as described above, were submitted to HPLC analysis.
High-performance liquid chromatography A Spectra Physics high-performance liquid chromatograph (model SP 8000) equipped with manual injector and ternary gradient system was used (Spectra Physics B.V., Eindhoven, The Netherlands). The stainless-steel column (250 × 4.6 mm i.d.) was packed with Spherisorb 10 ODS (Chrompack B.V., Middelburg, The Netherlands). The linear gradient was established with two solvents. Solvent A consisted of 0.5 M formic acid, 0.14 M pyridine (pH 3.0) and solvent B was l-propanol. All solvents were purified on a 0.45/am filter (type HA, Millipore B.V., Brussels, Belgium). Preliminary experiments were conducted concerning the gradient profile to optimize resolution of neurointermediate lobe peptides. The final gradient elution adopted, is included on the chromatograms. During the gradient elution the pressure increased from 700 to 2000 p.s.i. The polypeptides were concentrated on the head of the column during a 2-min loading phase. Equilibration of the column to the initial conditions lasted about 10 min. In all experiments, the sample volume brought on the column was 500/~1 of lobe extract. The column was eluted with a flow rate of 2 ml/min and 0.5-min fractions were collected with a fraction collector (LKB Redirac, model 2112). To each fraction 4 ml Aqua luma (Baker Chemicals B.V., Deventer, The Netherlands) was added and the samples were counted on a liquid scintillation analyser (Philips, model PW 4510). To determine the elution time of ct-MSH on the HPLCsystem, synthetic ct-MSH (Peninsula, San Carlos, California, USA) was dissolved in 250 #1 solvent A and detected in the eluate fractions at 280nm (Zeiss spectrophotometer, model PM 6).
Electrophoretic analysis To compare the results of the HPLC analysis with previous data of electrophoretic analysis, some of the labelled peptides separated by HPLC were submitted to electrophoresis. To the appropriate HPLC fractions, 50/~g BSA was added, the fractions were freeze-dried and then analysed by the acid-urea electrophoretic procedure described earlier (Jenks et al., 1979). It was evident that some of the peptides that were resolved by HPLC had not been found in the previous electrophoretic study (Jenks & van Overbeeke, 1980). As in this last investigation trichloroacetic acid (TCA) was used to prepare lobe homogenates, the possibility was considered that these new "HPLC peptides" constitute a TCAsoluble pool. A lobe extract from a pulse-chase incubation using [3H]lysine was divided into two equal aliquots. One aliquot was precipitated with 20?o TCA and the precipitate analysed with electrophoresis. The other aliquot was freeze-dried and the lyophilized extract was submitted to electrophoretic analysis.
RESULTS
H P L C analysis of pulse and pulse-chase incubations H P L C analysis of the incorporation of [3H]lysine into neurointermediate lobes of Xenopus laevis during the pulse period, showed one major labelled product (peak XI), eluting at 50 min (Fig. 1, filled circles). The peaks eluting in the first 10 min were [3H]lysine and contaminations in the [-3H]lysine preparation. After a 6-hr chase incubation, peak XI was greatly reduced and at least 12 other products (peaks I-X, XII and XIII) could be resolved on the reversed-phase column with the gradient system used (Fig. 1, open circles).
cpm.lO-) l-~'l( ) 39L'73663
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MSH bioassay Neurointermediate lobes were homogenized in 500 #1 0.1 M HAC, the homogenate was centrifuged, and the supernatant was submitted to HPLC. To the 1-min HPLC fractions, 50 #g BSA was added and the samples were freeze-dried. The Anolis carolinensis skin bioassay, as described by Tilders et al. (1975), was used with minor modifications. Each freeze-dried fraction was dissolved in 50/A A. carolinensis Ringer's solution and, as a preliminary survey for melanotropic activity, 20/~1 of this solution was added to 100/A Ringer's solution containing a small piece of A. carolinensis skin. Those fractions which showed melanotropic activity (darkening of the skin) were further diluted to determine their relative potency, i.e. the dilution factor whereby the sample was capable of visibly changing the bright green colour of the skin sample into a greenishbrown within 15 rain.
ACTH bioassay HPLC-fractions, 0.5min, were prepared as described above under "MSH bioassay". ACTH bioactivity of individual fractions was determined using adrenal cell suspensions following the Sayers assay (Sayers et al., 1971) in a modification as reported by Goverde et al. (1980). The sensitivity of this modified bioassay was approx 1 pg per ml cell suspension, using human ACTH 1-39 (CIBA-Geigy Ltd, Basel, Switzerland) as a standard.
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Fig. 1. High-performance liquid radiochromatogram of neurointermediate lobe peptides of black adapted Xenopus laevis. The tissue was pulse incubated for 30 min (filled circles) or pulse-chase incubated (30 min pulse, 6-hr chase; open circles) with [3H]lysine. Separation at ambient temperature was on Spherisorb 10 ODS (250 x 4.6 mm i.d.) with a flow rate of 2 ml/min; fractions were collected every 30 sec. Primary solvent (A): 0.5 M formic acid4).14 M pyridine (pH 3.0). Secondary solvent (B): 1-propanol.
495
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Fig. 2(a) Melanotropic activity of a high-performance liquid chromatogram prepared from neurointermediate lobe extract of black adapted Xenopus laevis. Activity of 1-min fractions was determined (see also Materials and Methods). (b) Adrenocorticotropic activity of a highperformance liquid chromatogram prepared from neurointermediate lobe extract of black adapted Xenopus laevis. Activity of 0.5-min fractions was determined (see also Materials and Methods). The chromatographic conditions were the same as given in Fig. 1.
It could be established that, following the chase period, only a very small amount of product XI was present in the incubation medium (data not shown). Release of newly synthesized products is currently under investigation.
Melanotropic and corticotropic activity Using the Anolis carolinensis skin bioassay it was shown that there were three major peaks of melanotropic activity (Fig. 2a). The elution times for these products corresponded exactly with those of chase peptides I, II and IV. Synthetic ct-MSH eluted from the HPLC column at 17 rain, which is between peaks II and IV (data not shown). The bioassay for adrenocorticotropic activity demonstrated the presence of a number of minor peaks and one major peak (Fig. 2b). The elution time of the major peak corresponded exactly with that of chase peptide II. Acid-urea gel electrophoresis The electrophoretic pattern for the freeze-dried extract of 30-min pulse, 6-hr chase incubated neurointermediate lobes is illustrated in Fig. 3 (open circles). This pattern is essentially the same as that reported by Jenks & van Overbeeke (1980) with the exception that there are three new products, namely W, Y and Z, which migrate to gel slices 42, 45-46 and 48 respectively. Analysis of the TCA precipitated lobe extract showed these peaks W, Y and Z to be absent from the electrophoretogram (Fig. 3, filled circles), which indicates that products W, Y and Z are TCAsoluble. Electrophoresis-~f HPLC-peaks I, II and IV,
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Fig. 3. Acid-urea gel electrophoresis of products synthesized in neuro-intermediate lobes of black adapted Xenopus laevis during a 30-min pulse, 6-hr chase incubation using [3H]lysine. Part of the lobe extract was freezedried (open circles). The remainder was TCA-precipitated; the precipitate produced essentially the same electrophoretogram, with the exception of the area between gel slices 40 and 50 (filled circles).
which corresponded to peaks of major bioactivity (Fig. 2), revealed that these products co-migrated with Z, Y and E respectively. Product VI appeared to comigrate with the TCA-soluble peptide W. The major labelled product of the pulse incubation, product XI, corresponded with product A on the acid-urea gel.
[ 3H] Leucine incorporation Comparison of the peak pattern following [3H]leucine incorporation (Fig. 4) with that after [3H]lysine
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496
GERARDJ. M. MARTENS,B. G. JENKSand A. P. VAN OVERBEEKE
incorporation (Fig. 1) strongly indicates that peaks I and II do not contain leucine. To improve the resolution of the area concerned, the same leucine-labelled sample was analysed using a less steep gradient (Fig. 4, insert). It was confirmed that products I and II, present after [3H]lysine incorporation, were absent from the chromatogram. DISCUSSION HPLC, in general, has some important advantages over electrophoretic techniques: (1) higher capacity for sample loading which no longer necessitates TCA precipitation or freeze-drying of the extract, (2) shorter separation time, (3) higher resolution and (4) possibility to adjust the resolution in selected areas. In using HPLC with a reversed-phase column, we did, however, experience one problem, namely that initially product XI did not elute from the column. In our opinion, this may have been due to a loading effect, such as irreversible binding to residual silanol groups. This suggestion was earlier proposed by O'Hare & Nice (1979) to explain the lack of elution of some high-molecular weight proteins from a reversedphase column. The fact that in our study after repeated analyses using the same column, peak XI did eventually elute, suggests that a conditioning of the column occurred, presumably through saturation of the silanol groups. The results of our HPLC analysis of pulse and pulse-chase incubations revealed the synthesis in the neurointermediate lobe of 13 products. Since peak XI represented the only major product during the pulse incubation and since this peak, furthermore, became reduced during the chase period, simultaneous with the appearance of the 12 other peaks, it may be concluded that product XI most likely functions as a precursor for the 12 chase peptides. This confirms the existence of a precursor-product mode of biosynthesis in the neurointermediate lobe, reported by Jenks & van Overbeeke (1980). It is therefore not surprising that product XI appeared to have the same electrophoretic properties as product A, considered to be the precursor by Jenks & van Overbeeke (1980). Three of the chase peptides, namely I, II and IV, had high melanotropic activity but, at least on the basis of elution time, none of these corresponded to c~-MSH. The lack of any newly synthesized peptide corresponding to c~-MSH seems to support the suggestion by Jenks & van Overbeeke (1980) that c~-MSH is not a major biosynthetic product in fully black adapted X e n o p u s laevis. The melanotropic peptides I and II are most likely the same products as peptides Z and Y, respectively, and they are, therefore, TCA-soluble. Moreover, on acid-urea gels these two peptides ran faster than c~-MSH (a-MSH migrates to gel slices 34-35, Jenks et al., 1979). Both their solubility in TCA and their electrophoretic behaviour point to the likelihood that peptides I and II are small peptides. The occurrence in X e n o p u s laevis of a TCAsoluble, melanotropic peptide has been reported earlier by Loh & Gainer (1977). The amino acid composition of the peptides I and II, discovered in our study, remains to be elucidated. The fact, however, that they have melanotropic activity indicates that they contain the "message sequence" ACTH 4 ~o
(Schwyzer & Eberle, 1977). Thus, they may well be structurally related to ~-MSH. The absence of the amino acid leucine in peptides I and II, too, is reminiscent of ~-MSH, since leucine is not found in any of the known ~-MSH structures. It is surprising that the major part of the corticotropic activity eluted with the small, TCA-soluble, melanotropic peptide II and not with any of the larger peptides. Schwyzer et al., (1971) demonstrated that small peptides, namely fragments of ACTH 1 24 possess some corticotropic activity, but far less than the complete molecule (ACTH 1 24). If, indeed, the corticotropic potency of peptide II were low, then peptide II must have been present in a high concentration. Alternatively, it might be a new, small peptide with a high corticotropic potency. The nature of the small bioactive peptides I and II is currently under investigation. Acknowledgements We thank Messrs H. J. M. Goverde and G. J. Pesman for performing the ACTH bioassays and Mrs E. M. Jansen-Hoorweg for expert typing.
REFERENCES
BUROERS A. (3. J. (196l) Occurrence of three electrophoretic components with melanocyte-stimulating activity in extracts of single pituitary glands from ungulates. Endocrinology 68, 698-703. GOVERDEH. J. M., PESMANG. J. & BENRAADTH. J. (1980) Improved sensitivity to adrenocorticotropin after purification and preincubation of rat adrenal cells. Acta Endocr. in press. HOPKINS C. R. (1972) The biosynthesis, intracellular transport, and packaging of melanocyte-stimulating peptides in the amphibian pars intermedia. J. Cell Biol. 53, 642-653. JENKS B. G., MEULEPASW. J. A. M., SOONSP. J. A. & VAN OVERBEEKE A. P. (1979) Biosynthesis of MSH and related peptides in the pars intermedia of the mouse: a pulse-chase analysis. Mol. cell. Endocr. 13, 149 158. JENKS B. G. & VAN OVERBEEKEA. P. (1980) Biosynthesis and release of neurointermediate lobe peptides in the aquatic toad, Xenopus laevis, adapted to a black background. Comp. Biochem. Physiol. 66, in press. Loll Y. P. & GAINER H. (1977) Biosynthesis, processing, and control of release of melanotropic peptides in the neurointermediate lobe of Xenopus laevis. J. gen. Physiol. 70, 37 58. LOll Y. P. (1979) Immunological evidence for two common precursors to corticotropins, endorphins, and melanotropins in the neurointermediate lobe of the toad pituitary. Proc. natn. Acad. Sci. U.S.A. 76, 796-800. NICE E. C. & O'HARE M. J. (1979) Simultaneous separation of ,8-1ipotrophin, adrenocorticotropic hormone, endorphins and enkephalins by high-performance liquid chromatography. J. Chromat. 162, 401-407. O'HARE M. J. & NICE E. C. (1979) Hydrophobic highperformance liquid chromatography of hormonal polypeptides and proteins on alkylsilane-bonded silica. J. Chromat. 171, 209 226. PRESLOCKJ. P. & BRINKLEYH. J. (1970a: Chemical quantification of melanophore stimulating substances using polyacrylamide gel disc-electrophoresis. Life Sci. 9, 215 -228. PRESLOCK J. P. & BRINKLEYH. J. (1970b) Melanophore and adrenocortical stimulating activities of substances from the pars intermedia, pars distalis, hypothalamus and cerebral cortex of the frog, Rana pipiens. Limb Sci. 9, 1369-1380.
Peptide synthesis in neurointermediate lobe of Xenopus laevis SAYERS G., SWALLOW R. L. & GIORDANO N. D. (1971) An improved technique for the preparation of isolated rat adrenal cells. A sensitive, accurate and specific method for the assay of ACTH. Endocrinology 88, 1063-1068. SCHWYZER R., SCHILLER P., SEELIG S. t~ SAYERSG. (1971) Isolated adrenal cells: log dose response curves for steroidogenesis induced by ACTH, 24, ACTH,_,o, ACTH4_lo, and ACTH 5 lo. FEBS Lett. 19, 229-231.
497
SCHWYZER R. & EBERLEA. (1977) On the molecular mechanism of ct-MSH receptor interactions. Front. Hormone Res. 4, 18-25. TILDERS F. J. H., VAN DELFT A. M. L. & SMELIK P. G. (1975) Re-introduction and evaluation of an accurate high capacity bioassay for melanocyte stimulating hormone using the skin of Anolis carolinensis in vitro. J. Endocr. 66, 165 175.