BIOCHEMICAL
Vol. 170, No. 2, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages
July 31, 1990
643- 648
ISOLATION OF A NEUROPEPTIDE CORRESPONDING TO THE N-TERMINAL 27 RE,SIDUES OF THE PITUITARY ADENYLATE CYCLASE ACTIVATING POLYPEPTIDE WITH 38 RESIDUES (PACAP38) Atsuro Miyata?, Lun Jiang, Raymond D. Dahl, Chieko Kitada*, Kazuki Kubo*, Masahiko Fujino*, Naoto Minamino? andAkira Arimuras U.S.-JapanBiomedicalResearchLaboratories,Tulane University Hcbert Center, Belle Chasse,LA 70037 Departmentsof Medicine, Physiology andAnatomy, TulaneUniversity School of Medicine, New Orleans,LA 70121 * TsukubaResearchLaboratories, Takeda ChemicalIndustries, Ltd., Tsukuba, Ibaraki 30042, Japan Received
June
8,
1990
SUMMARY: A novel neuropeptide with 38 residues(PACAP38) was isolated from ovine hypothalamictissuesusingthe pituitary adenylatecyclase activation in rat pituitary cell culturesas a parameterof the biological activity (Miyata et al, Biochem. Biophys. Res. Commun. 164, 567574, 1989). From the sidefractions obtainedduring the purification of PACAP38, a shorter form peptide with 27 residuescorresponding to the N-terminal 27 amino acids of PACAP and amidated C-terminus was isolated and named as PACAP27. Synthetic PACAP showed a biological activity of adenylatecyclasestimulation comparableto PACAP38. Moreover PACAP which showsa considerablehomology with vasoactive intestinalpolypeptide (VIP) demonstrateda similar vasodepressoractivity asVIP, but the adenylatecyclasestimulatingactivity was about 1000 timesgreater than VIP 01990Academic ETe**,Inc.
In an attempt to discover a novel hypothalamic hypophysiotropic hormone, a novel neuropeptidewith 38 residueswhich activates adenylate cyclase in rat pituitary cell cultures was isolated from ovine hypothalamic tissues,and named as pituitary adenylate cyclase activating polypeptide with 38 residues(PACAP38) (1). The N-terminal subsequence[l-28] of PACAP showed 68% homology with vasoactive intestinal polypeptide (VIP). cDNA of ovine PACAP precursor protein was subsequentlycloned, showing that preproPACAP38 is quite different from that of VIP and lacking of peptide histidine isoleucineamide(PHI) (2). Moreover, the biological activity of PACAP in termsof adenylatecyclase stimulating activity (ACSA) in rat pituitary cell cultures wasabout 1000timesgreater than that of VIP andcomparablewith corticotropin releasing factor (CRF) (1). During the purification of PACAP
in ovine hypothalamic extracts, the side
t Current Address: Department of Pharmacology, National Cardiovascular Center ResearchInstitute, Suita, Osaka565, Japan. 5 To whom all correspondenceshouldbe addressed. 0006-291x/90
643
$1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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fractions which showed considerable ACSA were purified and a shorter form of the peptide corresponding to the N-terminal 1-27 residues of PACAP was isolated. This paper describes isolation, chemical characterization, synthesis and the biological activity of this 27 residues neuropeptide which was named as PACAP27. MATERIALS
AND METHODS
Isolation: The starting material used to isolate PACAP was a side fraction obtained during the purification of PACAP from ovine hypothalamic tissues (1). Acetone-treated acid extracts of ovine hypothalamic tissues (approximately 2,400 g) were adsorbed on a C-18 silica column and eluted with step-wise increment (10, 20, 30, 40, 50 and 60%) of acetonitrile (CH3CN) in 0.1% trifluoroacetic acid (TFA). These fractions were designated as fr. A, B, C, D, E, and F, respectively. Fr. C which showed a marked ACSA was dissolved in 1 M acetic acid (AcOH) and placed on a SP-Sephadex C-25 column, and then eluted step-wise with 1 M AcOH (fr. SP-I), 2 M pyridine (fr. SP-II) and 2 M pyridine-acetate (pH 5.0) (fr. SP-III). Subsequent gel filtration of SPIII which showed a marked ACSA was performed on a column of Sephadex G-50 (fine, 5.5 x 97 cm) using 2 M AcOH containing 0.02% R-mercaptoethanol (R-ME) as the eluting solvent. A broad peak with ACSA corresponding to m.w. l,OOO-7,000 was eluted. The fractions with the greatest ACSA corresponding to m.w. 3,000-4,000 were pooled and lyophilized. The residue was reconstituted with 10 mM ammonium formate (pH 6.5) containing 10% CH3CN and adsorbed onto a CM52 cellulose column (1 x 38 cm, Whatman). Cation exchange chromatography was performed using a linear gradient clution of ammonium formate from 10 mM to 0.75 M as described previously (1). Fractions #37-43 which corresponded to the second greatest ACSA peak were used for the present purification. After desalting with Sep-Pak C-18 cartridge (Waters Associates), these fractions were submitted to reverse phase HPLC on a column of TSK ODS 120T (8.0 x 300 mm, Toyosoda) under the conditions detailed in the legend of Fig. la. The fractions with major ACSA were pooled and further purified by another reverse phase HPLC on a column of Vydac phenyl(4.6 x 250 mm, The Separations Group) (Fig. lb). The final purification was performed on a column of Delta Pak C-18 (3.9 x 150 mm, Waters Associates) (Fig. lc). Column effluents on HPLC were monitored by measuring the UV absorbance at 210 nm and 280 nm simultaneously. Amino acid and seauence analvsis: Amino acid analysis was carried out with Pica Tag system (Waters Associates) after acid hydrolysis of the highly purified peptide in 6 N HCL containing 0.1% phenol at 110°C for 24 h. Sequence analysis was performed by a pulse of liquid phase sequencer (Model 477A/ 120A, Applied Biosystcms). The resulting PTH-amino acids were analyzed by reverse phase HPLC in concert with the sequencer. To ascertain whether the carboxyterminal residue was present in an amidated or free acid form, the retention times of synthetic peptides of PACAP with an amidated and fret C-terminus were compared to that of the isolated peptide on microbore reverse phase HPLC system as detailed in Fig. 2. Synthesis of PACAP27: The 27 residue peptides with a C-terminal amide and free form according to the sequence result were synthesized by the solid phase technique, conducted on a 4-methylbenzhydrylamine resin and phenylacetamidemethyl resin, respectively. The synthetic products were purified successively by gel filtration on a Sephadex LH-20 column, ion exchange chromatography on a CM-cellulofine column and reverse phase HPLC on a Lichroprep RP-18 column. Correct synthesis was confirmed by amino acid analysis and mass spectrometry. Bioassay: ACSA was assessed by the accumulation of CAMP in rat pituitary cell culture media for the three-hr period after addition of a test material as described previously (1). Vasodepressor activity of the test material was determined in pentobarbital anesthetized male Wistar rats by monitoring the arterial blood pressure through the left carotid arterial canula by means of a pressure transducer (NEC San-Ei P23XL 45383) after a bolus injection of the peptide. The test sample was injected into the left femoral vein through the implanted cannula. RESULTS AND DISCUSSION As reported previousty, the CM cation exchange chromatography
of the ovine hypothalamic
extracts yielded three minor ACSA peaks in addition to the major peak. These peaks were eluted earlier than the major broad peak from which PACAP was isolated (1). The fractions 644
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I
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40
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b __--- -- _____________----. __________ __
a
10
20
40
30
50 I
0.4
c
------- _________
- ---- ----_/--_________
-60 g _ 5 --10 20
0.2
h1
0 0
10
I
I
I I
20
30
40
Retention
Time
50
(mid
Fie. la.First reverse phase HPLC of the ACSA fraction. Sample: half of the fractions 37-43 with the second greatest ACSAobtained by CM cation exchange chromatography (see Fig. 2 in ref. 1). Flow rate: 3.0 mlimin. Column: TSK ODS 120T (8.0 x 300 mm, ToyoSoda). Solvent system: linear gradient elution from A:B = 1OO:O to A:B = SO:20 for 6 min, followed by the second gradient from A:B = 80:20 to 35:65 for 54 min. (A) H20 : CH3CN : 10% TFA = 90 : 10 : 1 (v/v) (B) H20 : CH3CN : 10% TFA = 40 : 60 : 1 (v/v). The black bar shows the accumulation of CAMP during 3-hr incubation as the responce parameter of ACSA. Fip. 1b. Second reverse phase HPLC of the ACSA fraction. Sample: the major ACSA fraction eluted at 38.5-39.5 min in Fig. la. Flow rate: 1.0 ml/min. Column: Vydac phenyl (4.6 x 250 mm, The Separations Group). Solvent system: the same condition as above. Fi?. Ic.Final purification of PACAP by reverse phase HPLC. Sample: the ACSA fraction eluted at 36-36.5 min on reverse phase HPLC OCFig.lb. Flow rate: 1.0 mlimin. Column: Deltapak C-18 (3.9 x 150 mm, Waters Associates). Solvent system: the same condition as above.
correspondingto the secondgreatestACSA peak which appearedto be lessbasicthan PACAP was further purified by reversephaseHPLC on a TSK ODS 120T column. As shown in Fig. la, the prominent ACSA was eluted at 38539.5 min. These fractions were pooled and subjectedto another reverse phaseHPLC on a Vydac phenyl column (Fig. lb). The active fractions were finally reduced to purity by a Delta Pak C-18 column (Fig. 645
1~).
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Fip. 2a.b.Reverse phase HPLC of native PACAP and synthetic replicates containing either a free acid or an amidated carboxyl terminus. Sample: (a) coinjection of 40 ng of synthetic PACAP27NH2 and 40 ng of native PACAP27. (b) coinjection of 40 ng of synthetic PACAP27-OH and 40 ng of native PACAP27. Flow rate: 50 yl/min. Column: MAP310 ODS (1.0 x 100 mm, YMC Inc.). Solvent system: linear gradient elution from A:B = 1OO:O to A:B = 67:33 for 6 min, followed by the second gradient from A:B = 67:X to 25:75 for 60 min. (A) Hz0 : CH3CN : 10% TFA = 90 : 10 : 1 (v/v) (B) Hz0 : CH$N : 10% TFA = 40 : 60 : 1 (v/v). Arrows 1 and 2 indicates the elution positions of synthetic PACAP27-OH and PACAP27-NH2, respectively.
The amino acid composition
of this highly purified
peptide was : Asp; 1.81 [2], Glu; 1.02 [I],
Ser; 2.75 [3], Gly; 1.10 [l], His; 0.85 [l], Arg; 2.20 [2], Thr;
0.95 [l],
Ala; 3.21 [3], Tyr; 2.88
[3], Met; 0.94 [l], Phe; 1.00 [l], Leu; 2.13 [2], Ile; 1.01 [l], Val; 2.09 [2], Lys; 3.12 [3]. The peptide was sequenced
from 1 to 27 residues using 100 to 200 picomole
of the peptide.
The primary structure of the peptide was revealed as : His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Met-Ala-Val-Lys-LysTyr-Leu-Ala-Ala-Val-Leu-NH2. This sequence shown
was identical
with
in Fig. 2, the native peptide
terminus
that of the N-terminal co-&ted
with
but eluted separately from the peptide
Therefore,
the C-terminus
named as Pituitary
Cyclase Activating
Polypeptide
PACAP27. 646
residues
peptide
with a free C-terminus
of this native peptide was considered
Adenylate
l-27
the synthetic
of PACAP38.
with
As C-
on reverse phase HPLC.
to be amidated. with
an amidated
27 residues,
This peptide abbreviated
was as
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PACAP38:
His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Met-~a-Val-Lys
PACAP27:
His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Met-Ala-Val-Lys
VIP:
Hia-Ser-Asp-~-~-Phe-Thr-Asp-~-Tyr-~-~g-~-Arg-Lys-Gln-Met-Ala-val-Lys
21
30
-Lys-Tyr-L~u-~a-Al~-V~l-L~ufGly-Lys-Ar~Ty~-LyS-Gl~-A~g-V~l-Ly~-~~-Ly~-NH2 -Lys-Tyr-Leu-Rla-Ala-Val-Leu-NH2
w.The sequence comparison of PACAP with PACAP and VIP Residues which are underlined indicate amino acids different from those for PACAP27.
As shown in Fig. 3, PACAP possesses three pairs of basic amino acid residue. Lys29Arg30 is precededby Gly28, suggestingthe processingof PACAP by cleavage and amidationat this portion in a similar mannerto the other amidatedbioactive peptides(3). It should be noted that the C-terminusof PACAP is also amidated,thus casting a doubt that PACAP serves as the precursorof PACAP27. It is, however, possiblethat PACAP and PACAP could be cleaved independentlyat thesetwo different sitesof amidation from the PACAP precursor protein. This possibility hasbeensupportedby the finding that a radioimmunoassayspecific for the C-terminus of PACAP3S failed to detect the presenceof its C-terminal octapeptidein the ovine hypothalamic extract (unpublisheddata). If so, this may be the most unique feature of PACAP in the post-translationalprocessing, sincesuch an alternative amidation hasnever been observedin the processingof other bioactive peptidesso far identified. The final yield of PACAP
was about 350 pmole from 2,400 g of
60
04
-‘13
-12
-11
-10 Dose
(M) -9
-6
-7
-6
0 5
-
0.1
Dose
(nM)
1.0
w4. Adenylate cyclase stimulating activity of PACAP (0) as compared with those of PACAP (0) and VIP (A) determined using rat pituitary cell cultures as described in the text. u.Dose response curves of PACAP and VIP for arterial blood pressure in anesthetized male Wistar rats (n=5). Peptides (0.1, 0.3 and 1.0 nmole) were administered i.v. (a) and (0) indicate PACAP and VIP, respectively.
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ovine hypothalamic tissues, which corresponded to about one eighth the content of PACAP in the same tissues (1). The content of PACAP in the ovine hypothalamus was comparable to that of hypothalamic VIP (unpublished observation). It is conceivable that PACAP and PACAP27, both with an amidated C-terminus, are present in the ovine hypothalamic tissues as two mature forms. Whether PACAP study.
and PACAP
The ACSA of synthetic PACAP
play different physiological
roles warrants
was comparable with that of PACAP
further
in rat pituitary cell
cultures as shown in Fig. 4, suggesting that the region in the N-terminal l-27 residues is essential for the bioactivity. However, whether all these 27 residues are required for ACSA remains to be examined. Despite a considerable homology with VIP, ACSA of PACAP was about 1000 times greater than that of VIP It is worthwhile
to investigate which amino acids are critical for ACSA.
On the other hand, the rat hypothalamus and pituitary
membrane preparations contained high
affinity, specific binding sites for PACAP (4). These binding sites are shared with PACAP and PACAP27, but not by VIP (4), suggesting different physiological roles of PACAP from VIP in the pituitary and the central nervous system. As shown in Fig. 5, however, the vasodepressor VIP This suggests that PACAP immunohistochemical
activity of PACAP
was similar to that of
may also function as a vasoregulator similar to VIP Our recent
study with ovine brain indicated that some PACAP immunopositive
fibers
surrounded small blood vessels in the brain (5). This finding may also support a possible role of PACAP as a vasoregulatory
peptide. ACKNOWLEDGMENTS
This study was supported in part by NIH grant DK09094. We are grateful to Mrs. P. Portilla and M. C. Noriega for their technical assistance. REFERENCES 1) Miyata, A., Arimura, A., Dahl, R.D., Minamino, N., Uehara, A., Jiang, L., Culler M.D. and Coy D.H. (1989) Biochem. Biophys. Res. Commun. 164, 567-574. 2) Kimura, C., Ohkubo, S., Ogi, K., Hosoya, M., Itoh, Y., Onda, H., Miyata, A., Jiang, L., Dahl, R.D., Stibbs, H.H., Arimura, A. and Fujino, M. (1990) Biochem. Biophys. Res. Commun. 166, 81-89. 3) Eipper, B.A. & Mains, R.E. (1988) Ann. Rev. Physiol. 50, 333-344. 4) Gottschall, P.E., Tatsuno, I., Miyata, A. and Arimura, A. (1990) Endocrinology, in press. 5) Koves, K., Arimura, A., Somogyvari-Vigh, A., Vigh, S and Miller, J. (1990) Endocrinology, in press.
648
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643- 648
ISOLATION OF A NEUROPEPTIDE CORRESPONDING TO THE N-TERMINAL 27 RE,SIDUES OF THE PITUITARY ADENYLATE CYCLASE ACTIVATING POLYPEPTIDE WITH 38 RESIDUES (PACAP38) Atsuro Miyata?, Lun Jiang, Raymond D. Dahl, Chieko Kitada*, Kazuki Kubo*, Masahiko Fujino*, Naoto Minamino? andAkira Arimuras U.S.-JapanBiomedicalResearchLaboratories,Tulane University Hcbert Center, Belle Chasse,LA 70037 Departmentsof Medicine, Physiology andAnatomy, TulaneUniversity School of Medicine, New Orleans,LA 70121 * TsukubaResearchLaboratories, Takeda ChemicalIndustries, Ltd., Tsukuba, Ibaraki 30042, Japan Received
June
8,
1990
SUMMARY: A novel neuropeptide with 38 residues(PACAP38) was isolated from ovine hypothalamictissuesusingthe pituitary adenylatecyclase activation in rat pituitary cell culturesas a parameterof the biological activity (Miyata et al, Biochem. Biophys. Res. Commun. 164, 567574, 1989). From the sidefractions obtainedduring the purification of PACAP38, a shorter form peptide with 27 residuescorresponding to the N-terminal 27 amino acids of PACAP and amidated C-terminus was isolated and named as PACAP27. Synthetic PACAP showed a biological activity of adenylatecyclasestimulation comparableto PACAP38. Moreover PACAP which showsa considerablehomology with vasoactive intestinalpolypeptide (VIP) demonstrateda similar vasodepressoractivity asVIP, but the adenylatecyclasestimulatingactivity was about 1000 timesgreater than VIP 01990Academic ETe**,Inc.
In an attempt to discover a novel hypothalamic hypophysiotropic hormone, a novel neuropeptidewith 38 residueswhich activates adenylate cyclase in rat pituitary cell cultures was isolated from ovine hypothalamic tissues,and named as pituitary adenylate cyclase activating polypeptide with 38 residues(PACAP38) (1). The N-terminal subsequence[l-28] of PACAP showed 68% homology with vasoactive intestinal polypeptide (VIP). cDNA of ovine PACAP precursor protein was subsequentlycloned, showing that preproPACAP38 is quite different from that of VIP and lacking of peptide histidine isoleucineamide(PHI) (2). Moreover, the biological activity of PACAP in termsof adenylatecyclase stimulating activity (ACSA) in rat pituitary cell cultures wasabout 1000timesgreater than that of VIP andcomparablewith corticotropin releasing factor (CRF) (1). During the purification of PACAP
in ovine hypothalamic extracts, the side
t Current Address: Department of Pharmacology, National Cardiovascular Center ResearchInstitute, Suita, Osaka565, Japan. 5 To whom all correspondenceshouldbe addressed. 0006-291x/90
643
$1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
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No.
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fractions which showed considerable ACSA were purified and a shorter form of the peptide corresponding to the N-terminal 1-27 residues of PACAP was isolated. This paper describes isolation, chemical characterization, synthesis and the biological activity of this 27 residues neuropeptide which was named as PACAP27. MATERIALS
AND METHODS
Isolation: The starting material used to isolate PACAP was a side fraction obtained during the purification of PACAP from ovine hypothalamic tissues (1). Acetone-treated acid extracts of ovine hypothalamic tissues (approximately 2,400 g) were adsorbed on a C-18 silica column and eluted with step-wise increment (10, 20, 30, 40, 50 and 60%) of acetonitrile (CH3CN) in 0.1% trifluoroacetic acid (TFA). These fractions were designated as fr. A, B, C, D, E, and F, respectively. Fr. C which showed a marked ACSA was dissolved in 1 M acetic acid (AcOH) and placed on a SP-Sephadex C-25 column, and then eluted step-wise with 1 M AcOH (fr. SP-I), 2 M pyridine (fr. SP-II) and 2 M pyridine-acetate (pH 5.0) (fr. SP-III). Subsequent gel filtration of SPIII which showed a marked ACSA was performed on a column of Sephadex G-50 (fine, 5.5 x 97 cm) using 2 M AcOH containing 0.02% R-mercaptoethanol (R-ME) as the eluting solvent. A broad peak with ACSA corresponding to m.w. l,OOO-7,000 was eluted. The fractions with the greatest ACSA corresponding to m.w. 3,000-4,000 were pooled and lyophilized. The residue was reconstituted with 10 mM ammonium formate (pH 6.5) containing 10% CH3CN and adsorbed onto a CM52 cellulose column (1 x 38 cm, Whatman). Cation exchange chromatography was performed using a linear gradient clution of ammonium formate from 10 mM to 0.75 M as described previously (1). Fractions #37-43 which corresponded to the second greatest ACSA peak were used for the present purification. After desalting with Sep-Pak C-18 cartridge (Waters Associates), these fractions were submitted to reverse phase HPLC on a column of TSK ODS 120T (8.0 x 300 mm, Toyosoda) under the conditions detailed in the legend of Fig. la. The fractions with major ACSA were pooled and further purified by another reverse phase HPLC on a column of Vydac phenyl(4.6 x 250 mm, The Separations Group) (Fig. lb). The final purification was performed on a column of Delta Pak C-18 (3.9 x 150 mm, Waters Associates) (Fig. lc). Column effluents on HPLC were monitored by measuring the UV absorbance at 210 nm and 280 nm simultaneously. Amino acid and seauence analvsis: Amino acid analysis was carried out with Pica Tag system (Waters Associates) after acid hydrolysis of the highly purified peptide in 6 N HCL containing 0.1% phenol at 110°C for 24 h. Sequence analysis was performed by a pulse of liquid phase sequencer (Model 477A/ 120A, Applied Biosystcms). The resulting PTH-amino acids were analyzed by reverse phase HPLC in concert with the sequencer. To ascertain whether the carboxyterminal residue was present in an amidated or free acid form, the retention times of synthetic peptides of PACAP with an amidated and fret C-terminus were compared to that of the isolated peptide on microbore reverse phase HPLC system as detailed in Fig. 2. Synthesis of PACAP27: The 27 residue peptides with a C-terminal amide and free form according to the sequence result were synthesized by the solid phase technique, conducted on a 4-methylbenzhydrylamine resin and phenylacetamidemethyl resin, respectively. The synthetic products were purified successively by gel filtration on a Sephadex LH-20 column, ion exchange chromatography on a CM-cellulofine column and reverse phase HPLC on a Lichroprep RP-18 column. Correct synthesis was confirmed by amino acid analysis and mass spectrometry. Bioassay: ACSA was assessed by the accumulation of CAMP in rat pituitary cell culture media for the three-hr period after addition of a test material as described previously (1). Vasodepressor activity of the test material was determined in pentobarbital anesthetized male Wistar rats by monitoring the arterial blood pressure through the left carotid arterial canula by means of a pressure transducer (NEC San-Ei P23XL 45383) after a bolus injection of the peptide. The test sample was injected into the left femoral vein through the implanted cannula. RESULTS AND DISCUSSION As reported previousty, the CM cation exchange chromatography
of the ovine hypothalamic
extracts yielded three minor ACSA peaks in addition to the major peak. These peaks were eluted earlier than the major broad peak from which PACAP was isolated (1). The fractions 644
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0 I
II
1
IO
20
t
I
I
30
40
50
b __--- -- _____________----. __________ __
a
10
20
40
30
50 I
0.4
c
------- _________
- ---- ----_/--_________
-60 g _ 5 --10 20
0.2
h1
0 0
10
I
I
I I
20
30
40
Retention
Time
50
(mid
Fie. la.First reverse phase HPLC of the ACSA fraction. Sample: half of the fractions 37-43 with the second greatest ACSAobtained by CM cation exchange chromatography (see Fig. 2 in ref. 1). Flow rate: 3.0 mlimin. Column: TSK ODS 120T (8.0 x 300 mm, ToyoSoda). Solvent system: linear gradient elution from A:B = 1OO:O to A:B = SO:20 for 6 min, followed by the second gradient from A:B = 80:20 to 35:65 for 54 min. (A) H20 : CH3CN : 10% TFA = 90 : 10 : 1 (v/v) (B) H20 : CH3CN : 10% TFA = 40 : 60 : 1 (v/v). The black bar shows the accumulation of CAMP during 3-hr incubation as the responce parameter of ACSA. Fip. 1b. Second reverse phase HPLC of the ACSA fraction. Sample: the major ACSA fraction eluted at 38.5-39.5 min in Fig. la. Flow rate: 1.0 ml/min. Column: Vydac phenyl (4.6 x 250 mm, The Separations Group). Solvent system: the same condition as above. Fi?. Ic.Final purification of PACAP by reverse phase HPLC. Sample: the ACSA fraction eluted at 36-36.5 min on reverse phase HPLC OCFig.lb. Flow rate: 1.0 mlimin. Column: Deltapak C-18 (3.9 x 150 mm, Waters Associates). Solvent system: the same condition as above.
correspondingto the secondgreatestACSA peak which appearedto be lessbasicthan PACAP was further purified by reversephaseHPLC on a TSK ODS 120T column. As shown in Fig. la, the prominent ACSA was eluted at 38539.5 min. These fractions were pooled and subjectedto another reverse phaseHPLC on a Vydac phenyl column (Fig. lb). The active fractions were finally reduced to purity by a Delta Pak C-18 column (Fig. 645
1~).
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Fip. 2a.b.Reverse phase HPLC of native PACAP and synthetic replicates containing either a free acid or an amidated carboxyl terminus. Sample: (a) coinjection of 40 ng of synthetic PACAP27NH2 and 40 ng of native PACAP27. (b) coinjection of 40 ng of synthetic PACAP27-OH and 40 ng of native PACAP27. Flow rate: 50 yl/min. Column: MAP310 ODS (1.0 x 100 mm, YMC Inc.). Solvent system: linear gradient elution from A:B = 1OO:O to A:B = 67:33 for 6 min, followed by the second gradient from A:B = 67:X to 25:75 for 60 min. (A) Hz0 : CH3CN : 10% TFA = 90 : 10 : 1 (v/v) (B) Hz0 : CH$N : 10% TFA = 40 : 60 : 1 (v/v). Arrows 1 and 2 indicates the elution positions of synthetic PACAP27-OH and PACAP27-NH2, respectively.
The amino acid composition
of this highly purified
peptide was : Asp; 1.81 [2], Glu; 1.02 [I],
Ser; 2.75 [3], Gly; 1.10 [l], His; 0.85 [l], Arg; 2.20 [2], Thr;
0.95 [l],
Ala; 3.21 [3], Tyr; 2.88
[3], Met; 0.94 [l], Phe; 1.00 [l], Leu; 2.13 [2], Ile; 1.01 [l], Val; 2.09 [2], Lys; 3.12 [3]. The peptide was sequenced
from 1 to 27 residues using 100 to 200 picomole
of the peptide.
The primary structure of the peptide was revealed as : His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Met-Ala-Val-Lys-LysTyr-Leu-Ala-Ala-Val-Leu-NH2. This sequence shown
was identical
with
in Fig. 2, the native peptide
terminus
that of the N-terminal co-&ted
with
but eluted separately from the peptide
Therefore,
the C-terminus
named as Pituitary
Cyclase Activating
Polypeptide
PACAP27. 646
residues
peptide
with a free C-terminus
of this native peptide was considered
Adenylate
l-27
the synthetic
of PACAP38.
with
As C-
on reverse phase HPLC.
to be amidated. with
an amidated
27 residues,
This peptide abbreviated
was as
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PACAP38:
His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Met-~a-Val-Lys
PACAP27:
His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Met-Ala-Val-Lys
VIP:
Hia-Ser-Asp-~-~-Phe-Thr-Asp-~-Tyr-~-~g-~-Arg-Lys-Gln-Met-Ala-val-Lys
21
30
-Lys-Tyr-L~u-~a-Al~-V~l-L~ufGly-Lys-Ar~Ty~-LyS-Gl~-A~g-V~l-Ly~-~~-Ly~-NH2 -Lys-Tyr-Leu-Rla-Ala-Val-Leu-NH2
w.The sequence comparison of PACAP with PACAP and VIP Residues which are underlined indicate amino acids different from those for PACAP27.
As shown in Fig. 3, PACAP possesses three pairs of basic amino acid residue. Lys29Arg30 is precededby Gly28, suggestingthe processingof PACAP by cleavage and amidationat this portion in a similar mannerto the other amidatedbioactive peptides(3). It should be noted that the C-terminusof PACAP is also amidated,thus casting a doubt that PACAP serves as the precursorof PACAP27. It is, however, possiblethat PACAP and PACAP could be cleaved independentlyat thesetwo different sitesof amidation from the PACAP precursor protein. This possibility hasbeensupportedby the finding that a radioimmunoassayspecific for the C-terminus of PACAP3S failed to detect the presenceof its C-terminal octapeptidein the ovine hypothalamic extract (unpublisheddata). If so, this may be the most unique feature of PACAP in the post-translationalprocessing, sincesuch an alternative amidation hasnever been observedin the processingof other bioactive peptidesso far identified. The final yield of PACAP
was about 350 pmole from 2,400 g of
60
04
-‘13
-12
-11
-10 Dose
(M) -9
-6
-7
-6
0 5
-
0.1
Dose
(nM)
1.0
w4. Adenylate cyclase stimulating activity of PACAP (0) as compared with those of PACAP (0) and VIP (A) determined using rat pituitary cell cultures as described in the text. u.Dose response curves of PACAP and VIP for arterial blood pressure in anesthetized male Wistar rats (n=5). Peptides (0.1, 0.3 and 1.0 nmole) were administered i.v. (a) and (0) indicate PACAP and VIP, respectively.
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No.
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BIOCHEMICAL
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ovine hypothalamic tissues, which corresponded to about one eighth the content of PACAP in the same tissues (1). The content of PACAP in the ovine hypothalamus was comparable to that of hypothalamic VIP (unpublished observation). It is conceivable that PACAP and PACAP27, both with an amidated C-terminus, are present in the ovine hypothalamic tissues as two mature forms. Whether PACAP study.
and PACAP
The ACSA of synthetic PACAP
play different physiological
roles warrants
was comparable with that of PACAP
further
in rat pituitary cell
cultures as shown in Fig. 4, suggesting that the region in the N-terminal l-27 residues is essential for the bioactivity. However, whether all these 27 residues are required for ACSA remains to be examined. Despite a considerable homology with VIP, ACSA of PACAP was about 1000 times greater than that of VIP It is worthwhile
to investigate which amino acids are critical for ACSA.
On the other hand, the rat hypothalamus and pituitary
membrane preparations contained high
affinity, specific binding sites for PACAP (4). These binding sites are shared with PACAP and PACAP27, but not by VIP (4), suggesting different physiological roles of PACAP from VIP in the pituitary and the central nervous system. As shown in Fig. 5, however, the vasodepressor VIP This suggests that PACAP immunohistochemical
activity of PACAP
was similar to that of
may also function as a vasoregulator similar to VIP Our recent
study with ovine brain indicated that some PACAP immunopositive
fibers
surrounded small blood vessels in the brain (5). This finding may also support a possible role of PACAP as a vasoregulatory
peptide. ACKNOWLEDGMENTS
This study was supported in part by NIH grant DK09094. We are grateful to Mrs. P. Portilla and M. C. Noriega for their technical assistance. REFERENCES 1) Miyata, A., Arimura, A., Dahl, R.D., Minamino, N., Uehara, A., Jiang, L., Culler M.D. and Coy D.H. (1989) Biochem. Biophys. Res. Commun. 164, 567-574. 2) Kimura, C., Ohkubo, S., Ogi, K., Hosoya, M., Itoh, Y., Onda, H., Miyata, A., Jiang, L., Dahl, R.D., Stibbs, H.H., Arimura, A. and Fujino, M. (1990) Biochem. Biophys. Res. Commun. 166, 81-89. 3) Eipper, B.A. & Mains, R.E. (1988) Ann. Rev. Physiol. 50, 333-344. 4) Gottschall, P.E., Tatsuno, I., Miyata, A. and Arimura, A. (1990) Endocrinology, in press. 5) Koves, K., Arimura, A., Somogyvari-Vigh, A., Vigh, S and Miller, J. (1990) Endocrinology, in press.
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