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Proportions of mono- and diacetylated forms of alpha MSH in individual neurointermediate lobe extracts of Cyprinus carpio L.
Comp. Biochem. Physiol. Vol. 84B, No. 3, pp. 315-317, 1986 Printed in Great Britain
0305-0491/86 $3.00 + 0.00 Pergamon Journals Ltd
PROPORTIONS OF MONO- A N D DIACETYLATED FORMS OF ALPHA MSH IN INDIVIDUAL N E U R O I N T E R M E D I A T E LOBE E X T R A C T S OF C Y P R I N U S C A R P I O L. E. FOLLf~NIUS*,A. VAN DORSSELAER~"and A. MEUNIER Laboratoire de Cytologie Animale, and #Laboratoire de Chimie des Substances Naturelles (Centre de Neurochimie): Universit6 Louis Pasteur, Strasbourg, France (Received 18 December 1985) The proportions of the mono- and diacetylated forms of ctMSH in individual carp neurointermediate lobe (NIL) extracts, as assessed by HPLC and RIA, fluctuate within a narrow range (mono/mono- + diacetyl ctMSH = 3-14%). 2. These results obtained on individuals of different sex and age show that the diacetylated form predominates in the NIL of all individuals where it represents in the mean 90% of the acetylated forms. 3. The relatively low fluctuation of the proportions of the acetylated forms suggests that the possibility for a physiological modulation of the diacetylation step may be limited in this species of fish.
Abstract--1.
In recent studies on the melanocyte-stimulating hormone of different vertebrates it has been demonstrated that the desacetylated, mono- and diacetylated forms coexist in the intragranular pool of the intermediate lobe cells. Soon after the first studies made on mammals ( R u d m a n et al., 1979; Buckley et al., 1981; Dell et al., 1982; G o l d m a n et al., 1983) the same forms have been isolated and partially characterized by H P L C , R I A and mass spectrometry in two species of cyprinid fishes (Follrnius et al., 1985). F o r the moment the physiological significance of the acetylation process is very poorly understood. The N-acetylation enhances the melanocyte-stimulating potency of ~tM S H (Eberl6 et al., 1978; Sumpter et al., 1984). Only one comparative study concerning the melanocyte-stimulating and the lipolytic activities of the mono- and diacetylated forms is available now (Rudman et al., 1983). It may thus be of interest to consider more closely the proportions of the monoacetylated and diacetylated forms because they result from two acetylation steps which occur during the biosynthesis of ctMSH. One may wonder whether the two forms are present in about the same proportion throughout the year without relation to the glandular activity or inversely whether the ratio varies according to physiological parameters to be defined. In a first approach we consider the range of variation of the ratio m o n o / ( m o n o + diacetylated)alpha M S H as it is established from H P L C separations from homogenates Of individual carp neurointermediate lobes.
High-performance liquid chromatography and RIA Detailed information on the chromatographic system used has already been published (Follrnius et al., 1985). Briefly: the supernatant of the NIL homogenate is injected on a Waters C 18 Bondapak column and eluted by a gradient of two solutions: (A) 20% methanol, 80% water, 1% acetic acid and 0.05% triethylamine; (B) 80% methanol, 20% water, 1% acetic acid and 0.05% triethylamine. Elution began at 20% B and reached a final 73% B on a concave gradient No. 7. The fractions previously identified as containing the forms of ctMSH were collected in the glass tubes, lyophilized in a Speed-Vac system, recovered in 1 ml of RIA buffer and assayed with the antiserum used in the previous studies. For the RIA the fractions were diluted to 1/100-I/1000 to fall within the working range (5-125 pg) of our RIA. The detection limit (B/Bo = 90%) was 5 pg/tube and the mean sensitivity (B/Bo = 50%) 26 pg. All samples were tested in triplicate. Data about the specificity of the antiserum have already been given (Follrnius et al., 1985). Our results document also a good accuracy of the whole technology applied in our laboratories for the separation and quantification of the forms of ~tMSH.
MATERIALS AND METHODS
RESULTS
To assess the degree of acetylation of the intracellular ~MSH the previously published techniques were applied *Correspondence to be addressed to E. Follenius, 12 Rue de l'Universitr, 67000 Strasbourg, France. 315 C.B.P, 84/31~-F
(Follrnius et al., 1985). The sensitivity of the RIA suffices for an accurate estimation of the MSH content of HPLC fractions of individual neurointermediate lobes (NIL). Four groups of carp purchased from local sources were used in the present study. To eliminate other hypophyseal sources of POMC derived peptides, the NIL lobes were dissected in situ and transferred to vials containing 500/zl ice cold 1% acetic acid with 500 KIU/ml of "Trasylol". After a short sonication (30 sec), cell debris was removed by centrifugation at 4°C and the supernatant was frozen at -20°C until the chromatographic separations.
To illustrate the good reproducibility of the H P L C chromatograms of individual N I L homogenates four chromatograms are given in Fig. 1. They belong to individuals of different sex and of different weight (Table 1). There is a close similarity of the major
316
E. FOLLI~NIUSet al. .~750
DO
1
2R0n rr
2~
i
• =7,=
,1500 .1250 .1000 -750 -500 -250
'1750 DO 280~
DO 2B0n n"
-15CXJ ,1250 .1000 7'50
~-zso )8
s
20
2S
3b
Fig. 1. HPLC chromatograms of NIL homogenates from carp (series 212 a, c, d, e). The RIA results of fractions 15, 22, 25 are superimposed on each graph. X scale: fraction number; left Y scale optical density in arbitrary units; right Y scale: quantity of immunoreactive ~MSH/fraction in ng/tube. (1) Elution position of standard desacetyl MSH; (2) elution of standard position MSH (3) elution position of diacetyl ~MSH. peaks especially o f those which c o r r e s p o n d to the • M S H forms. T h e three previously identified forms: the desacetyl-, monoacetyl-, a n d diacetyl ~ M S H are present. The respective height of the c o r r e s p o n d i n g peaks is of similar amplitude which illustrates the fact also s u p p o r t e d by the subsequent R I A ' s t h a t only very small differences in the p r o p o r t i o n s of the different forms occur in different individuals. T o quantify more closely the different forms the c o r r e s p o n d i n g fractions were assayed for i m m u n o reactive c~M S H forms. T h e results are s u m m a r i z e d in Table 1. The study o f the content o f individual N I L provides i n f o r m a t i o n which is usually n o t accessible t h r o u g h R I A ' s of batches of several NIL. The assessm e n t of the different forms of ~ M S H s was m a d e o n a n individual basis to establish the p r o p o r t i o n s of the acetylated forms. T h e range of v a r i a t i o n o f the
p r o p o r t i o n s o f A / A + B is from 3.2 to 14% with a m e a n of 9.9%. This range includes also the sampling a n d assay errors• DISCUSSION The results of this study have to be considered from several points of view• First they confirm the high degree o f acetylation of the M S H sequence stored in the N I L cells. F r o m i m m u n o c y t o c h e m i c a l studies (Stoeckel et al., 1983) m a d e o n the IL cells of the r a b b i t it was s h o w n t h a t the M S H s are stored in the secretory granules, where the N-acetylation process is achieved. In Xenopus the N-acetylation is considered to be the final step of the posttranslational processing, which m e a n s t h a t it m a y be delayed until the excretion is initiated. G o l d m a n a n d
Table 1. Proportions of mono- and diacetylated aMSH in individual NILs of Cyprinus carpio L. Date of Desacetyl Monoacetyl Diacetyl A Ind kill Sex W e i g h t aMSH (D) ~MSH (A)* aMSH (B)* A + B% a 20.9.84 ~ 750 g N.D. 23 690 3.2 Series 207 b 20.9.84 ~ 1000 N.D. 40 630 6 a 21.9.84 ~ 650 N.D. 73 513 14 Series 208 b 21.9.84 ~ 450 N.D. 78 690 10 c 21.9.84 ~ 540 N.D. 51 655 7.2 a 21.11.84 ~ 350 N.D. 68 520 11 Series 211 b 21.11.84 ~ 350 N.D. 40 720 5 c 21.11.84 ~ 450 N.D. 135 820 14 d 21.11.84 ~ 700 N.D. 153 957 13 a 15.1.85 ~ 1100 22 198 1920 9 Series 212 b 15.1.85 ~ 900 22 78 500 13 c 15.1.85 9 950 23 140 1190 10 d 15.1.85 ~ 1100 5 65 640 9 e 15.1.85 ~ 800 7.5 86 662 11 720 ± 265 15 ± 8 87 ± 50 793 ± 372 9.9 Mean ± SD *In ng/LNI.
Acetylated MSHs in carp NIL Loh (1984) consider however that this secretionlinked acetylation is probably only a minor process; the main pathway being secretion independent. In this species the modulation of the acetylation in relation with the background adaptation is postulated (Jenks et al., 1985). From our results one may conclude that in the carp the post-translational acetylations are nearly complete before excretion. Almost all the MSH is found under its biacetylated form. About 10% of the acetylated etMSH is monoacetylated. No data are now available to discuss the possibility of an artefactual desacetylation during the preparative steps. Precautions, such as manipulation at low temperature during the initial steps and adjunction of Trasylol have been taken to minimize them. For the moment it is not established that all the forms detected in the homogenates originate in the intermediate lobe cells, even if this is the most likely hypothesis. A possible contribution of the neural lobe component cannot be a priori excluded. The high degree of double acetylation of the ctMSH sequence can no longer be considered as specific of the intermediate lobe tissue. A similar process has also been described in the mammalian pancreas, where 78% of the MSH is diacetylated (Hirsch and O'Donohue, 1985). The assessment of the content of individual NIL's for the different forms shows that the proportions of the acetylated forms are nearly the same regardless of the absolute values. The fish considered in the present study were of different sex and of different size and age and they were maintained under normal conditions (12L/D) during the 3 days preceding killing. Under these conditions the proportions of the acetylated forms in the intragranular pool are relatively stable. This may result from a well-defined equilibrium of these forms in the granules, but numerous other explanations may be considered. More data are needed about the post-translational acetylation steps and the specific storage conditions at the molecular level before a pertinent explanation of the stability of the proportions of the different acetylated forms may be given. As far as we are aware no data are now available concerning individual fluctuations of the proportions of the acetylated forms in the NIL of other vertebrates. Goldman and Loh (1984) consider only the N-acetylation step in X e n o p u s in relation to physiological and experimental parameters since, as shown earlier (Martens et al., 1981) in this species the post-translational acetylation seems to be restricted to the N-acetylation which may be modulated (Jenks et aL, 1985) in relation to the background adaptation process. Similar studies have now to be made on fish to establish whether the different acetylation steps are modulated in relation to background adaptation. The physiological meaning of the diacetylation has still to be established. In mammals (Rudman et al., 1983) no clear difference in the melanotropic potency of the mono- and diacetylated ~ MSH was found. The main difference was an increase of the biological half life of the circulating diacetylated form as compared with the monoaeetylated one. As shown recently (Follrnius et al., accepted) both forms are excreted and are circulating in the blood. In the carp, their proportions there do not match the proportions
317
found in the NIL cells. One may therefore wonder whether both acetylated forms have the same role and whether the diacetylated form has to be desacetylated to generate the physiologically active monoacetylated form. In theory both forms may be physiologically active provided specific receptors exist on the corresponding target cells.
REFERENCES
Buckley D. L., Houghten R. A. and Ramachandran J. (1981) Isolation of melanotropin and N-O diacetylserine ct melanotrotropin from porcine pituitary extracts. Int. J. Pept. Prot. Res. 17, 508-513. Dell A., Etienne T., Panico M., Morris H. R., Vinson G., Whitehouse B., Barber M., Bodoli R. S., Edgewick R. and Tyler A. N. (1982) Characterization of an adrenal glomerulosa-stimulating component of posterior pituitary extract as biacetyl-Ser 1 ~MSH. Neuropeptides (Edinburgh) 2, 233-240. Ebert6 A., Chang Y. and Schwyzer R. (1978) Chemical synthesis and biological activity of the dogfish (Squalus acanthias) ct melanotropins I and II, and of related peptides. Heir. chim. Acta 61, 2360-2374. Follrnius E., Van Dorsselaer A. and Meunier A. (1985) Separation and partial characterization by highperformance liquid chromatography and radioimmunoassay of different forms of melanocytestimulating hormone from fish (Cyprinidae) neurointermediate lobes. Gen. comp. Endocr. 57, 198-207. Follrnius E., Van Dorsselaer A. and Meunier A. Circulating forms of 0tMSH in the carp and trout blood. An HPLC and RIA study. Gen. comp. Endocr., accepted for publication. Hirsch M. D. and O'Donohue T. L. (1985) Characterization of alpha-melanocyte-stimulating hormone in rat pancreas. Peptides 6, 293-296. Goldman M. E., Beaulieu M., Kebabian J. W. and Eskay R. L. (1983) Melanocyte-stimulating hormone-like peptides in the intermediate lobe of the rat pituitary gland: characterization of content and release in vitro. Endocrinology, Buc. 112, 435-441. Goldman M. E. and Loh Y. P. (1984) Intracellular acetylation of desacetyl ~tMSH in the Xenopus laevis neurointermediate lobe. Peptides 5, 1129-I 134. Jenks B. G., Verburg-van Kemenade B. M. L. and Vaudry H. (1985) Regulation of N-terminal acetylation of MSH during background adaptations in the amphibian, Xenopus laevis. Thirteenth Annual Meeting for Biochemical Endocrinology, Edinburgh, September 1985 Abstr: p 20. Martens G. J. M., Jenks B. G. and Van Overbeeke A. P. (1981) N-acetylation is linked to ctMSH release from pars intermedia of amphibian pituitary gland. Nature, Lond. 294, 558-560. Rudman D., Chawla R. K. and Hollins B. M. (1979) N-O Diacetyl serine ct melanocyte stimulating hormone: a naturally occurring melanotropic peptide. J. biol. Chem. 254, 10102-10108. Rudman D., Hollins B. M., Kutner M. H., Moffit S. D. and Lynn M. J. (1983) Three types of melanostimulating hormones. Bioactivities and half-lives. Am. J. Physiol. 245, E47-E54. Stoeckel M. E., Shimchowitsch S., Garraud J. C., Schmitt G., Vaudry H. and Porte A. (1983) Immunocytochemical evidence of intragranular acetylation of ctMSH in the melanotropic cells of the rabbit. Cell Tiss. Res. 230, 511-515. Sumpter J. P., Denning-Kendall P. A. and Lowry P. J. (1984) The involvement of melanotrophins in physiological colour changes in the dogfish Scyliorhinus canicula. Gen. comp. Endocr. 56, 360-367.
0305-0491/86 $3.00 + 0.00 Pergamon Journals Ltd
PROPORTIONS OF MONO- A N D DIACETYLATED FORMS OF ALPHA MSH IN INDIVIDUAL N E U R O I N T E R M E D I A T E LOBE E X T R A C T S OF C Y P R I N U S C A R P I O L. E. FOLLf~NIUS*,A. VAN DORSSELAER~"and A. MEUNIER Laboratoire de Cytologie Animale, and #Laboratoire de Chimie des Substances Naturelles (Centre de Neurochimie): Universit6 Louis Pasteur, Strasbourg, France (Received 18 December 1985) The proportions of the mono- and diacetylated forms of ctMSH in individual carp neurointermediate lobe (NIL) extracts, as assessed by HPLC and RIA, fluctuate within a narrow range (mono/mono- + diacetyl ctMSH = 3-14%). 2. These results obtained on individuals of different sex and age show that the diacetylated form predominates in the NIL of all individuals where it represents in the mean 90% of the acetylated forms. 3. The relatively low fluctuation of the proportions of the acetylated forms suggests that the possibility for a physiological modulation of the diacetylation step may be limited in this species of fish.
Abstract--1.
In recent studies on the melanocyte-stimulating hormone of different vertebrates it has been demonstrated that the desacetylated, mono- and diacetylated forms coexist in the intragranular pool of the intermediate lobe cells. Soon after the first studies made on mammals ( R u d m a n et al., 1979; Buckley et al., 1981; Dell et al., 1982; G o l d m a n et al., 1983) the same forms have been isolated and partially characterized by H P L C , R I A and mass spectrometry in two species of cyprinid fishes (Follrnius et al., 1985). F o r the moment the physiological significance of the acetylation process is very poorly understood. The N-acetylation enhances the melanocyte-stimulating potency of ~tM S H (Eberl6 et al., 1978; Sumpter et al., 1984). Only one comparative study concerning the melanocyte-stimulating and the lipolytic activities of the mono- and diacetylated forms is available now (Rudman et al., 1983). It may thus be of interest to consider more closely the proportions of the monoacetylated and diacetylated forms because they result from two acetylation steps which occur during the biosynthesis of ctMSH. One may wonder whether the two forms are present in about the same proportion throughout the year without relation to the glandular activity or inversely whether the ratio varies according to physiological parameters to be defined. In a first approach we consider the range of variation of the ratio m o n o / ( m o n o + diacetylated)alpha M S H as it is established from H P L C separations from homogenates Of individual carp neurointermediate lobes.
High-performance liquid chromatography and RIA Detailed information on the chromatographic system used has already been published (Follrnius et al., 1985). Briefly: the supernatant of the NIL homogenate is injected on a Waters C 18 Bondapak column and eluted by a gradient of two solutions: (A) 20% methanol, 80% water, 1% acetic acid and 0.05% triethylamine; (B) 80% methanol, 20% water, 1% acetic acid and 0.05% triethylamine. Elution began at 20% B and reached a final 73% B on a concave gradient No. 7. The fractions previously identified as containing the forms of ctMSH were collected in the glass tubes, lyophilized in a Speed-Vac system, recovered in 1 ml of RIA buffer and assayed with the antiserum used in the previous studies. For the RIA the fractions were diluted to 1/100-I/1000 to fall within the working range (5-125 pg) of our RIA. The detection limit (B/Bo = 90%) was 5 pg/tube and the mean sensitivity (B/Bo = 50%) 26 pg. All samples were tested in triplicate. Data about the specificity of the antiserum have already been given (Follrnius et al., 1985). Our results document also a good accuracy of the whole technology applied in our laboratories for the separation and quantification of the forms of ~tMSH.
MATERIALS AND METHODS
RESULTS
To assess the degree of acetylation of the intracellular ~MSH the previously published techniques were applied *Correspondence to be addressed to E. Follenius, 12 Rue de l'Universitr, 67000 Strasbourg, France. 315 C.B.P, 84/31~-F
(Follrnius et al., 1985). The sensitivity of the RIA suffices for an accurate estimation of the MSH content of HPLC fractions of individual neurointermediate lobes (NIL). Four groups of carp purchased from local sources were used in the present study. To eliminate other hypophyseal sources of POMC derived peptides, the NIL lobes were dissected in situ and transferred to vials containing 500/zl ice cold 1% acetic acid with 500 KIU/ml of "Trasylol". After a short sonication (30 sec), cell debris was removed by centrifugation at 4°C and the supernatant was frozen at -20°C until the chromatographic separations.
To illustrate the good reproducibility of the H P L C chromatograms of individual N I L homogenates four chromatograms are given in Fig. 1. They belong to individuals of different sex and of different weight (Table 1). There is a close similarity of the major
316
E. FOLLI~NIUSet al. .~750
DO
1
2R0n rr
2~
i
• =7,=
,1500 .1250 .1000 -750 -500 -250
'1750 DO 280~
DO 2B0n n"
-15CXJ ,1250 .1000 7'50
~-zso )8
s
20
2S
3b
Fig. 1. HPLC chromatograms of NIL homogenates from carp (series 212 a, c, d, e). The RIA results of fractions 15, 22, 25 are superimposed on each graph. X scale: fraction number; left Y scale optical density in arbitrary units; right Y scale: quantity of immunoreactive ~MSH/fraction in ng/tube. (1) Elution position of standard desacetyl MSH; (2) elution of standard position MSH (3) elution position of diacetyl ~MSH. peaks especially o f those which c o r r e s p o n d to the • M S H forms. T h e three previously identified forms: the desacetyl-, monoacetyl-, a n d diacetyl ~ M S H are present. The respective height of the c o r r e s p o n d i n g peaks is of similar amplitude which illustrates the fact also s u p p o r t e d by the subsequent R I A ' s t h a t only very small differences in the p r o p o r t i o n s of the different forms occur in different individuals. T o quantify more closely the different forms the c o r r e s p o n d i n g fractions were assayed for i m m u n o reactive c~M S H forms. T h e results are s u m m a r i z e d in Table 1. The study o f the content o f individual N I L provides i n f o r m a t i o n which is usually n o t accessible t h r o u g h R I A ' s of batches of several NIL. The assessm e n t of the different forms of ~ M S H s was m a d e o n a n individual basis to establish the p r o p o r t i o n s of the acetylated forms. T h e range of v a r i a t i o n o f the
p r o p o r t i o n s o f A / A + B is from 3.2 to 14% with a m e a n of 9.9%. This range includes also the sampling a n d assay errors• DISCUSSION The results of this study have to be considered from several points of view• First they confirm the high degree o f acetylation of the M S H sequence stored in the N I L cells. F r o m i m m u n o c y t o c h e m i c a l studies (Stoeckel et al., 1983) m a d e o n the IL cells of the r a b b i t it was s h o w n t h a t the M S H s are stored in the secretory granules, where the N-acetylation process is achieved. In Xenopus the N-acetylation is considered to be the final step of the posttranslational processing, which m e a n s t h a t it m a y be delayed until the excretion is initiated. G o l d m a n a n d
Table 1. Proportions of mono- and diacetylated aMSH in individual NILs of Cyprinus carpio L. Date of Desacetyl Monoacetyl Diacetyl A Ind kill Sex W e i g h t aMSH (D) ~MSH (A)* aMSH (B)* A + B% a 20.9.84 ~ 750 g N.D. 23 690 3.2 Series 207 b 20.9.84 ~ 1000 N.D. 40 630 6 a 21.9.84 ~ 650 N.D. 73 513 14 Series 208 b 21.9.84 ~ 450 N.D. 78 690 10 c 21.9.84 ~ 540 N.D. 51 655 7.2 a 21.11.84 ~ 350 N.D. 68 520 11 Series 211 b 21.11.84 ~ 350 N.D. 40 720 5 c 21.11.84 ~ 450 N.D. 135 820 14 d 21.11.84 ~ 700 N.D. 153 957 13 a 15.1.85 ~ 1100 22 198 1920 9 Series 212 b 15.1.85 ~ 900 22 78 500 13 c 15.1.85 9 950 23 140 1190 10 d 15.1.85 ~ 1100 5 65 640 9 e 15.1.85 ~ 800 7.5 86 662 11 720 ± 265 15 ± 8 87 ± 50 793 ± 372 9.9 Mean ± SD *In ng/LNI.
Acetylated MSHs in carp NIL Loh (1984) consider however that this secretionlinked acetylation is probably only a minor process; the main pathway being secretion independent. In this species the modulation of the acetylation in relation with the background adaptation is postulated (Jenks et al., 1985). From our results one may conclude that in the carp the post-translational acetylations are nearly complete before excretion. Almost all the MSH is found under its biacetylated form. About 10% of the acetylated etMSH is monoacetylated. No data are now available to discuss the possibility of an artefactual desacetylation during the preparative steps. Precautions, such as manipulation at low temperature during the initial steps and adjunction of Trasylol have been taken to minimize them. For the moment it is not established that all the forms detected in the homogenates originate in the intermediate lobe cells, even if this is the most likely hypothesis. A possible contribution of the neural lobe component cannot be a priori excluded. The high degree of double acetylation of the ctMSH sequence can no longer be considered as specific of the intermediate lobe tissue. A similar process has also been described in the mammalian pancreas, where 78% of the MSH is diacetylated (Hirsch and O'Donohue, 1985). The assessment of the content of individual NIL's for the different forms shows that the proportions of the acetylated forms are nearly the same regardless of the absolute values. The fish considered in the present study were of different sex and of different size and age and they were maintained under normal conditions (12L/D) during the 3 days preceding killing. Under these conditions the proportions of the acetylated forms in the intragranular pool are relatively stable. This may result from a well-defined equilibrium of these forms in the granules, but numerous other explanations may be considered. More data are needed about the post-translational acetylation steps and the specific storage conditions at the molecular level before a pertinent explanation of the stability of the proportions of the different acetylated forms may be given. As far as we are aware no data are now available concerning individual fluctuations of the proportions of the acetylated forms in the NIL of other vertebrates. Goldman and Loh (1984) consider only the N-acetylation step in X e n o p u s in relation to physiological and experimental parameters since, as shown earlier (Martens et al., 1981) in this species the post-translational acetylation seems to be restricted to the N-acetylation which may be modulated (Jenks et aL, 1985) in relation to the background adaptation process. Similar studies have now to be made on fish to establish whether the different acetylation steps are modulated in relation to background adaptation. The physiological meaning of the diacetylation has still to be established. In mammals (Rudman et al., 1983) no clear difference in the melanotropic potency of the mono- and diacetylated ~ MSH was found. The main difference was an increase of the biological half life of the circulating diacetylated form as compared with the monoaeetylated one. As shown recently (Follrnius et al., accepted) both forms are excreted and are circulating in the blood. In the carp, their proportions there do not match the proportions
317
found in the NIL cells. One may therefore wonder whether both acetylated forms have the same role and whether the diacetylated form has to be desacetylated to generate the physiologically active monoacetylated form. In theory both forms may be physiologically active provided specific receptors exist on the corresponding target cells.
REFERENCES
Buckley D. L., Houghten R. A. and Ramachandran J. (1981) Isolation of melanotropin and N-O diacetylserine ct melanotrotropin from porcine pituitary extracts. Int. J. Pept. Prot. Res. 17, 508-513. Dell A., Etienne T., Panico M., Morris H. R., Vinson G., Whitehouse B., Barber M., Bodoli R. S., Edgewick R. and Tyler A. N. (1982) Characterization of an adrenal glomerulosa-stimulating component of posterior pituitary extract as biacetyl-Ser 1 ~MSH. Neuropeptides (Edinburgh) 2, 233-240. Ebert6 A., Chang Y. and Schwyzer R. (1978) Chemical synthesis and biological activity of the dogfish (Squalus acanthias) ct melanotropins I and II, and of related peptides. Heir. chim. Acta 61, 2360-2374. Follrnius E., Van Dorsselaer A. and Meunier A. (1985) Separation and partial characterization by highperformance liquid chromatography and radioimmunoassay of different forms of melanocytestimulating hormone from fish (Cyprinidae) neurointermediate lobes. Gen. comp. Endocr. 57, 198-207. Follrnius E., Van Dorsselaer A. and Meunier A. Circulating forms of 0tMSH in the carp and trout blood. An HPLC and RIA study. Gen. comp. Endocr., accepted for publication. Hirsch M. D. and O'Donohue T. L. (1985) Characterization of alpha-melanocyte-stimulating hormone in rat pancreas. Peptides 6, 293-296. Goldman M. E., Beaulieu M., Kebabian J. W. and Eskay R. L. (1983) Melanocyte-stimulating hormone-like peptides in the intermediate lobe of the rat pituitary gland: characterization of content and release in vitro. Endocrinology, Buc. 112, 435-441. Goldman M. E. and Loh Y. P. (1984) Intracellular acetylation of desacetyl ~tMSH in the Xenopus laevis neurointermediate lobe. Peptides 5, 1129-I 134. Jenks B. G., Verburg-van Kemenade B. M. L. and Vaudry H. (1985) Regulation of N-terminal acetylation of MSH during background adaptations in the amphibian, Xenopus laevis. Thirteenth Annual Meeting for Biochemical Endocrinology, Edinburgh, September 1985 Abstr: p 20. Martens G. J. M., Jenks B. G. and Van Overbeeke A. P. (1981) N-acetylation is linked to ctMSH release from pars intermedia of amphibian pituitary gland. Nature, Lond. 294, 558-560. Rudman D., Chawla R. K. and Hollins B. M. (1979) N-O Diacetyl serine ct melanocyte stimulating hormone: a naturally occurring melanotropic peptide. J. biol. Chem. 254, 10102-10108. Rudman D., Hollins B. M., Kutner M. H., Moffit S. D. and Lynn M. J. (1983) Three types of melanostimulating hormones. Bioactivities and half-lives. Am. J. Physiol. 245, E47-E54. Stoeckel M. E., Shimchowitsch S., Garraud J. C., Schmitt G., Vaudry H. and Porte A. (1983) Immunocytochemical evidence of intragranular acetylation of ctMSH in the melanotropic cells of the rabbit. Cell Tiss. Res. 230, 511-515. Sumpter J. P., Denning-Kendall P. A. and Lowry P. J. (1984) The involvement of melanotrophins in physiological colour changes in the dogfish Scyliorhinus canicula. Gen. comp. Endocr. 56, 360-367.