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Structure of the pentapeptide proctolin, a proposed neurotransmitter in insects
Life Sciences Vol . 17, pp . Printed in the U.S .A .
Pergamon Press
1253-1256
STRUCTURE OF THE PENTAPEPTIDE PROCTOLIN, A PROPOSED NEUROTRANSMITTER IN INSECTS Alvin N . Starratt and Brian E . Brown Research Institute, Agriculture Canada University Sub P. O . London, Canada N6A 5B7 (Received in final form September 15, 1975) Summary Proctolin, a myotropic substance with potent activity on the proctodeal (hindqut) muscles of the cockroach, Periplaneta americana (L .), has been identified by the use of Edman degra dation and dansylation techniques as Arg-Tyr-Leu-Pro-Thr. Synthesis of the pentapeptide having this sequence and exhibiting the properties of natural proctolin confirmed the structure. Threshold activity on proctodeal muscle occurs at about 10-9 M proctolin. In 1967, Brown presented preliminary evidence that a myotropic substance extracted fran the viscera of the cockroach, Periplaneta americana (L .), might function as an excitatory neurotransmitter in the visceral muscles of insects (1) . The active compound was stated to be none of the established or putative transmitters including glutamate (1), the proposed excitatory mediator in insect skeletal fibres (2) . Subsequently, HoLnan and Cook (3) claimed that the excitatory factor was a mixture of glutamate, aspartate, and a third component later reported to be a small, basic peptide (4) . They suggested that glutamate was the likely mediator (3) and that the peptide was a neurosecretory substance involved in the regulation of excitability (4) . The preceding report (5) suggests that insects possess an ultrastructurally distinct type of motor nerve cell in which transmission is mediated by a small peptide, proctolin, the myotropic substance of the initial study (1) . The isolation of proctolin has now been completed arr3 is detailed elsewhere (6) . The present communication reports the sequential analysis of proctolin and confirmation of the structure bY synthesis . Materials and Methods Proctolin was isolated from the cockroach, Periplaneta americana (L .) . Approximately 180 ug of proctolin were recovered from 125,000 insects (125 kg fresh weight) . The isolated proctolin was homogeneous by paper and thin-layer chranatography and by high voltage paper electrophoresis at pH 3.5 and 6.4 (6) . Determination of Structure : For the determination of amino acid composition, 2-4 ug quantities of proctolin were hydrolyzed by heating for 16 hr at 110° with 50 ul of constant-boiling HC1 in small sealed tubes. The hydro lysis products were dansylated (7) and chromatographed two-dimensionally on polyamide-6 (Macherey-Nagel) precoated sheets with water-90% formic acid (100 :1 .5) in the first direction and benzene-acetic acid (9 :1) in the second 1253
1254
Structure of Proctolin
Vol . 17, No . 8
direction and on silica gel F-254 (E . Merck) precoated plates with benzenepyridine-acetic acid (80:20 ;5) followed by toluene-2-chloroethanol-cons . ammonium hydroxide (6 :10;4) . The dansyl amino acids were located under W light. Their identity was confirmed by co-chromatography with small quantities of the dansyl derivatives of 14C-labelled amino acids which were detected by autoradiography . The amino acid sequence of proctolin was established by the Edman-dansyl method essentially as described by Gray and Smith (7) . The time of hydrolysis was normally 16 hr but was reduced to 6 hr for dansyl praline . Dansyl amino acids were identified as described above. In addition to determining the amino-terminal group at each cycle of degradation, the amino acid composition of the shortened peptide was also determined . Synthesis : Proctolin was synthesized in a stepwise fashion by the mixed anhydride procedure using isobutyl chloroformate, N-methylmorpholine, and suitably protected amino acids (B) . The pentapeptide derivative, Z-Arg(N02) Tyr(Bzl)-Leu-Pro-Thr-~1e, obtained in this manner was hydrolyzed and the remaining protecting groups removed by hydrogenolysis . The synthetic peptide was purified by ion-exchange chromatography (Rexyn 101, NHq form) and gel filtration (Sephadex G-15) as employed in the isolation of proctolin (6) . Complete details of the synthesis will be reported separately . Com orison of Natural and S nthetic Proctolin: Small quantities of natural and synthe c proctolin were chromatographed on paper and on silica gel and Avicel plates . High voltage paper electropherograms were obtained at pH 3.5 and 6.4 . Details of the solvent systems and buffers are presented in Table 1. Synthetic and natural proctolin were detected on chromatograms and electropherograms by ni.nhydrin spray or by assay on the isolated hindqut (1) ; in the latter case, small quantities of paper, Avicel or silica gel were added directly to the organ bath . Pharmacological tests on isolated proctodeal muscle (1,5) were performed with the synthetic and natural peptides to compare their specific activities, dose-response effects, and receptor agonist-antagonist relationships . The relative potencies of some closely related synthetic analogues of proctolin were also determined . Results and Discussion Several ninhydrin positive spots were obtained when acid hydrolysates or leucine aminopeptidase digests of proctolin were chroanatographed on paper, indi cating that the active substance was a peptide. Proctolin, as well as one of the hydrolysis products, reacted positively with Sakaguchi reagent suggesting the presence of arginine . The W absorption spectcvm of proctolin indicated the presence of tyrosine . Following acid hydrolysis and dansylation, thin-layer chromatograms showed spots corresponding to arginine, leucine, praline, threonine, and tyrosine in approximately equimolar amounts . Sequence analysis by the Edman-dansyl method indicated the following structure for proctolin: Arg-Tyr-Leu-Pro-Thr The structure was confirmed by synthesis of the peptide having this sequence by the mixed anhydride procedure. During purification by ion-exchange chromatography and gel filtration, the synthetic peptide was obtained at elu tion volumes identical with those of proctolin. The natural and synthetic peptides co-chranatographed on paper, and on Avicel and Kieselgel plates, and co-migrated upon high voltage paper electrophoresis at pH 3.5 and 6.4 (Table 1).
1255
Structure of Proctolin
Vol. 17, No . 8
TABLE 1
Chromatographic and Electrorophoresic Comparison of Natural and Synthetic Proctolin Chromatography Rf of Proctolin Sorbent Silica gel Avicel Paper
Solvent" A B A
High Voltage Paper Electrophoresis T~'ffer"" A B
pH
3.5 6.4
Natural 0 .17 0 .29 0 .45
Synthetic 0 .18 0 .29 0.47
Mixture 0 .18 0.30 0.46
Çathodal Mi~a tion of Proctolin (cm) Natural Synthetic Mixture 28 .5 20 .9
27 .6 20 .7
28 .2 21 .0
" Solvent A : butanol-acetic acid-water, 4:1 :5 . Solvent B: butanolacetic acid-water, 4 :1 :1 . "" Buffer A : pyridine-acetic acid-water, 1:10;445 . Buffer H: pyridine-acetic acid-water, 25 :1 :350 . Pharmacological assay danonstrated that the specific activity of the synthetic product was identical to that of the natural peptide and, furthermore, that the dose-response curves were parallel (Fig . lA) . As with Proctolin, the hindgut responses to the synthetic peptide were equally inhibited by 6 x 10 -7 M tyramine (Fig . 1B) . Fig . 1C demonstrates that equivalent but subthreshold quantities of both natural and synthetic Proctolin caused identical potentiation of neurally evoked contractions . Thus, on the basis of their chromatographic, electrophoretic, and pharmacological properties, the synthetic and natural peptides are indist in~+ishable. Proctolin (M .W . 648) is active on cockroach proctodeal muscle at threshold concentrations of about 10 -9 M. Preliminary results with a number of closely related synthetic analogues indicate that threonine is essential for full acti vity . Rsnoval of threonine or replacement by serine or by threonine amide led to substances with less than 1% of the activity of Proctolin. Rsnoval of arginine also resulted in a substantial loss of activity . These results further support the assigned structure of Proctolin. Holman and Cook (4) partially purified a small, basic peptide from the hindgut of another cockroach, Leucophaea maderae (Fabr.) and reported its presence in P . americana and the grasshopper, Schistocerca va a va a (Scudder) . Based on itsdescribed characteristics, this "hindqut stimulating peptide" is probably identical to Proctolin (6) . However, in contrast to the reported situation in Leucophaea (4), we find that Proctolin is both present in and active on the visceral muscle of the foregut of Periplaneta. Holman and Cook also report that their peptide appears to be restricted to the Orthoptera . We have extracted and partially processed representatives of the Orthoptera, Haniptera, Coleoptera, Lepidoptera, Diptera, and Hymenoptera. Each of the 8 species processed yielded a substance with pharmacological activity similar to that of Proctolin. In every instance, the chromatographic and electrophoretic behaviour of the active compound was identical to that of Proctolin . These results provide a substantive basis for proposing that Proctolin is a universal constituent of the Insecta . It is increasingly apparent that certain peptides of neural origin have significant functions in the vertebrate nervous systen, either as transmitter substances or as modulators of neuronal activity (9) . Proctolin is the first such peptide to be characterized in the Insecta and with the exception of a
1256
Structure of Proctolin
Syath~tie
Natural
t 0" 8
t I "2
Vol . 17, No . 8
t
~ 0~6
I "!
~ Iß 2 wla
f l 0~1 Syn
t 1 0"I Nat
t 1
0"2 Syn FIC~ . 1
t 1
0 " 2 ny / ml Nat
Canparative pharmacological assay of natural and synthetic proctolin on isolated proctodeal muscle of Periplaneta . A- Nozmal responses to equal quantities of the natural and synthetic peptide applied for 20 sec . B - Same doses applied to the same preparation in the presence of 6x10-7 M tyramine . C- Potentiation of neurally evoked contractions(BHz for 3 sec) by subthreshold concentrations of synthetic and natural proctolin . crustacean neurohormone (10), we believe it to be the first in invertebrates . The availability of synthetic proctolin will greatly facilitate the definition of its function as a neurohormone (4),or nrurotransmitter (1,5) . Acknowledgments : The authors wish to acknowledge the assistance of Mrs . M . E . Stevens, Mr . K . Bertrim and Mr . N . Jerry . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 .
B . E . BROWN, Science 155, 595-597 (1967) . P . N . R . USHERWOOD, Neûrosci . Res . Prod. Bull . 10, 136-143 (1972) . G . M . HOLMAN and 8 . J . COOK, J . Insect Ph siol .16, 1891-1907 (1970) . G . M . HOIMAN and 8 . J . COOK, Biol . Bull . 142, 44460 (1972) . B . E . BROWN, Life Sci . 17, 12252 1975) . B . E . BROWN and A .N . SThRRATT, J . Insect ph siol . in press . W . R . GRAY and J . F . SMITH, Anal . Biochor . 33~-42 (1970) . G . W . ANDERSON, J . E . ZIIß~RMAN, and F . M, CÂLIAHAN, _J . Amer . Chor . Soc . 89, 5012-5017 (1967) . L. L . IVII2SEN, Nature 252, 630 (1974) . P . FERNLUND and L . JOSEFSSON, Science 177, 173-175 (1972) .
Pergamon Press
1253-1256
STRUCTURE OF THE PENTAPEPTIDE PROCTOLIN, A PROPOSED NEUROTRANSMITTER IN INSECTS Alvin N . Starratt and Brian E . Brown Research Institute, Agriculture Canada University Sub P. O . London, Canada N6A 5B7 (Received in final form September 15, 1975) Summary Proctolin, a myotropic substance with potent activity on the proctodeal (hindqut) muscles of the cockroach, Periplaneta americana (L .), has been identified by the use of Edman degra dation and dansylation techniques as Arg-Tyr-Leu-Pro-Thr. Synthesis of the pentapeptide having this sequence and exhibiting the properties of natural proctolin confirmed the structure. Threshold activity on proctodeal muscle occurs at about 10-9 M proctolin. In 1967, Brown presented preliminary evidence that a myotropic substance extracted fran the viscera of the cockroach, Periplaneta americana (L .), might function as an excitatory neurotransmitter in the visceral muscles of insects (1) . The active compound was stated to be none of the established or putative transmitters including glutamate (1), the proposed excitatory mediator in insect skeletal fibres (2) . Subsequently, HoLnan and Cook (3) claimed that the excitatory factor was a mixture of glutamate, aspartate, and a third component later reported to be a small, basic peptide (4) . They suggested that glutamate was the likely mediator (3) and that the peptide was a neurosecretory substance involved in the regulation of excitability (4) . The preceding report (5) suggests that insects possess an ultrastructurally distinct type of motor nerve cell in which transmission is mediated by a small peptide, proctolin, the myotropic substance of the initial study (1) . The isolation of proctolin has now been completed arr3 is detailed elsewhere (6) . The present communication reports the sequential analysis of proctolin and confirmation of the structure bY synthesis . Materials and Methods Proctolin was isolated from the cockroach, Periplaneta americana (L .) . Approximately 180 ug of proctolin were recovered from 125,000 insects (125 kg fresh weight) . The isolated proctolin was homogeneous by paper and thin-layer chranatography and by high voltage paper electrophoresis at pH 3.5 and 6.4 (6) . Determination of Structure : For the determination of amino acid composition, 2-4 ug quantities of proctolin were hydrolyzed by heating for 16 hr at 110° with 50 ul of constant-boiling HC1 in small sealed tubes. The hydro lysis products were dansylated (7) and chromatographed two-dimensionally on polyamide-6 (Macherey-Nagel) precoated sheets with water-90% formic acid (100 :1 .5) in the first direction and benzene-acetic acid (9 :1) in the second 1253
1254
Structure of Proctolin
Vol . 17, No . 8
direction and on silica gel F-254 (E . Merck) precoated plates with benzenepyridine-acetic acid (80:20 ;5) followed by toluene-2-chloroethanol-cons . ammonium hydroxide (6 :10;4) . The dansyl amino acids were located under W light. Their identity was confirmed by co-chromatography with small quantities of the dansyl derivatives of 14C-labelled amino acids which were detected by autoradiography . The amino acid sequence of proctolin was established by the Edman-dansyl method essentially as described by Gray and Smith (7) . The time of hydrolysis was normally 16 hr but was reduced to 6 hr for dansyl praline . Dansyl amino acids were identified as described above. In addition to determining the amino-terminal group at each cycle of degradation, the amino acid composition of the shortened peptide was also determined . Synthesis : Proctolin was synthesized in a stepwise fashion by the mixed anhydride procedure using isobutyl chloroformate, N-methylmorpholine, and suitably protected amino acids (B) . The pentapeptide derivative, Z-Arg(N02) Tyr(Bzl)-Leu-Pro-Thr-~1e, obtained in this manner was hydrolyzed and the remaining protecting groups removed by hydrogenolysis . The synthetic peptide was purified by ion-exchange chromatography (Rexyn 101, NHq form) and gel filtration (Sephadex G-15) as employed in the isolation of proctolin (6) . Complete details of the synthesis will be reported separately . Com orison of Natural and S nthetic Proctolin: Small quantities of natural and synthe c proctolin were chromatographed on paper and on silica gel and Avicel plates . High voltage paper electropherograms were obtained at pH 3.5 and 6.4 . Details of the solvent systems and buffers are presented in Table 1. Synthetic and natural proctolin were detected on chromatograms and electropherograms by ni.nhydrin spray or by assay on the isolated hindqut (1) ; in the latter case, small quantities of paper, Avicel or silica gel were added directly to the organ bath . Pharmacological tests on isolated proctodeal muscle (1,5) were performed with the synthetic and natural peptides to compare their specific activities, dose-response effects, and receptor agonist-antagonist relationships . The relative potencies of some closely related synthetic analogues of proctolin were also determined . Results and Discussion Several ninhydrin positive spots were obtained when acid hydrolysates or leucine aminopeptidase digests of proctolin were chroanatographed on paper, indi cating that the active substance was a peptide. Proctolin, as well as one of the hydrolysis products, reacted positively with Sakaguchi reagent suggesting the presence of arginine . The W absorption spectcvm of proctolin indicated the presence of tyrosine . Following acid hydrolysis and dansylation, thin-layer chromatograms showed spots corresponding to arginine, leucine, praline, threonine, and tyrosine in approximately equimolar amounts . Sequence analysis by the Edman-dansyl method indicated the following structure for proctolin: Arg-Tyr-Leu-Pro-Thr The structure was confirmed by synthesis of the peptide having this sequence by the mixed anhydride procedure. During purification by ion-exchange chromatography and gel filtration, the synthetic peptide was obtained at elu tion volumes identical with those of proctolin. The natural and synthetic peptides co-chranatographed on paper, and on Avicel and Kieselgel plates, and co-migrated upon high voltage paper electrophoresis at pH 3.5 and 6.4 (Table 1).
1255
Structure of Proctolin
Vol. 17, No . 8
TABLE 1
Chromatographic and Electrorophoresic Comparison of Natural and Synthetic Proctolin Chromatography Rf of Proctolin Sorbent Silica gel Avicel Paper
Solvent" A B A
High Voltage Paper Electrophoresis T~'ffer"" A B
pH
3.5 6.4
Natural 0 .17 0 .29 0 .45
Synthetic 0 .18 0 .29 0.47
Mixture 0 .18 0.30 0.46
Çathodal Mi~a tion of Proctolin (cm) Natural Synthetic Mixture 28 .5 20 .9
27 .6 20 .7
28 .2 21 .0
" Solvent A : butanol-acetic acid-water, 4:1 :5 . Solvent B: butanolacetic acid-water, 4 :1 :1 . "" Buffer A : pyridine-acetic acid-water, 1:10;445 . Buffer H: pyridine-acetic acid-water, 25 :1 :350 . Pharmacological assay danonstrated that the specific activity of the synthetic product was identical to that of the natural peptide and, furthermore, that the dose-response curves were parallel (Fig . lA) . As with Proctolin, the hindgut responses to the synthetic peptide were equally inhibited by 6 x 10 -7 M tyramine (Fig . 1B) . Fig . 1C demonstrates that equivalent but subthreshold quantities of both natural and synthetic Proctolin caused identical potentiation of neurally evoked contractions . Thus, on the basis of their chromatographic, electrophoretic, and pharmacological properties, the synthetic and natural peptides are indist in~+ishable. Proctolin (M .W . 648) is active on cockroach proctodeal muscle at threshold concentrations of about 10 -9 M. Preliminary results with a number of closely related synthetic analogues indicate that threonine is essential for full acti vity . Rsnoval of threonine or replacement by serine or by threonine amide led to substances with less than 1% of the activity of Proctolin. Rsnoval of arginine also resulted in a substantial loss of activity . These results further support the assigned structure of Proctolin. Holman and Cook (4) partially purified a small, basic peptide from the hindgut of another cockroach, Leucophaea maderae (Fabr.) and reported its presence in P . americana and the grasshopper, Schistocerca va a va a (Scudder) . Based on itsdescribed characteristics, this "hindqut stimulating peptide" is probably identical to Proctolin (6) . However, in contrast to the reported situation in Leucophaea (4), we find that Proctolin is both present in and active on the visceral muscle of the foregut of Periplaneta. Holman and Cook also report that their peptide appears to be restricted to the Orthoptera . We have extracted and partially processed representatives of the Orthoptera, Haniptera, Coleoptera, Lepidoptera, Diptera, and Hymenoptera. Each of the 8 species processed yielded a substance with pharmacological activity similar to that of Proctolin. In every instance, the chromatographic and electrophoretic behaviour of the active compound was identical to that of Proctolin . These results provide a substantive basis for proposing that Proctolin is a universal constituent of the Insecta . It is increasingly apparent that certain peptides of neural origin have significant functions in the vertebrate nervous systen, either as transmitter substances or as modulators of neuronal activity (9) . Proctolin is the first such peptide to be characterized in the Insecta and with the exception of a
1256
Structure of Proctolin
Syath~tie
Natural
t 0" 8
t I "2
Vol . 17, No . 8
t
~ 0~6
I "!
~ Iß 2 wla
f l 0~1 Syn
t 1 0"I Nat
t 1
0"2 Syn FIC~ . 1
t 1
0 " 2 ny / ml Nat
Canparative pharmacological assay of natural and synthetic proctolin on isolated proctodeal muscle of Periplaneta . A- Nozmal responses to equal quantities of the natural and synthetic peptide applied for 20 sec . B - Same doses applied to the same preparation in the presence of 6x10-7 M tyramine . C- Potentiation of neurally evoked contractions(BHz for 3 sec) by subthreshold concentrations of synthetic and natural proctolin . crustacean neurohormone (10), we believe it to be the first in invertebrates . The availability of synthetic proctolin will greatly facilitate the definition of its function as a neurohormone (4),or nrurotransmitter (1,5) . Acknowledgments : The authors wish to acknowledge the assistance of Mrs . M . E . Stevens, Mr . K . Bertrim and Mr . N . Jerry . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 .
B . E . BROWN, Science 155, 595-597 (1967) . P . N . R . USHERWOOD, Neûrosci . Res . Prod. Bull . 10, 136-143 (1972) . G . M . HOLMAN and 8 . J . COOK, J . Insect Ph siol .16, 1891-1907 (1970) . G . M . HOIMAN and 8 . J . COOK, Biol . Bull . 142, 44460 (1972) . B . E . BROWN, Life Sci . 17, 12252 1975) . B . E . BROWN and A .N . SThRRATT, J . Insect ph siol . in press . W . R . GRAY and J . F . SMITH, Anal . Biochor . 33~-42 (1970) . G . W . ANDERSON, J . E . ZIIß~RMAN, and F . M, CÂLIAHAN, _J . Amer . Chor . Soc . 89, 5012-5017 (1967) . L. L . IVII2SEN, Nature 252, 630 (1974) . P . FERNLUND and L . JOSEFSSON, Science 177, 173-175 (1972) .