0013-?227/79/1056-1410$02.00/0 Endocrinology Copyright © 1979 by The Endocrine Society
Vol. 105, No. 6 Printed in U.S.A.
Isolation and Characterization of Somatostatin from Anglerfish Pancreatic Islet* BRYAN D. NOE,f JOACHIM SPIESS, JEAN E. RIVIER, AND WYLIE VALE Department of Anatomy, Emory University School of Medicine, Atlanta, Georgia 30322 and Marine Biological Laboratory, Woods Hole, Massachusetts 02543 (B.D.N.); and the Peptide Biology Laboratory at The Salk Institute for Biological Studies (J.S., J.E.R. and W. V.), La Jolla, California 92037
ABSTRACT. Somatostatin was purified from anglerfish pancreatic islets using acetic acid extraction, gel filtration (Bio-Gel P-10), ion exchange chromatography (CM Bio-Gel A), and reversed phase high pressure liquid chromatography. The resulting peptide was characterized by RIA, bioassay, and determination of amino acid composition. Anglerfish islet somatostatin was found to possess an amino acid composition and immunological
S
OMATOSTATIN (SRIF) has been localized by bioassay and/or immunological techniques in numerous tissues (1). It is not known, however, whether the SRIF-like species identified in all of these sites are identical in structure and biological activity. To date, only the primary structures of SRIF from ovine hypothalamus (2, 3), porcine hypothalamus (4), and pigeon pancreas (5) have been determined. SRIF isolated from each of these sources was found to have the same primary structure. It has been demonstrated recently that the biosynthesis of pancreatic islet SRIF involves a precursor-product pathway (6, 7). As part of an investigative effort designed to isolate and characterize the biosynthetic precursor for anglerfish islet SRIF (AF SRIF), we have purified and characterized tetradecapeptide SRIF from this source. We report here the results of this work. Materials and Methods
Materials Anglerfish islets (25 g) were obtained at the Marine Biological Laboratory (Woods Hole, MA), the Northeast Fisheries Center of the National Marine Fisheries Service (Woods Hole, MA), Received December 21, 1978. Address all correspondence and requests for reprints to: Dr. Bryan D. Noe, Department of Anatomy, Emory University School of Medicine, Atlanta, Georgia 30322. * This work was supported in part by NIH Grants AM-19890 and AM-16921 (to B.D.N.) and AM-20917 and AM-18811, National Foundation Grant 1-411, and a grant from the Texas Research Foundation/ Salk Institute (Peptide Biology Lab). f Recipient of a USPHS Research Career Development Award AM00142.
and biological activities equivalent to synthetic somatostatin. Sequence analyses revealed that the primary structure was HAla-Gly-cyclo-[Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-ThrSer-Cys]-OH. These results demonstrate that anglerfish islet somatostatin has the same primary structure as somatostatin from all other sources characterized to date. (Endocrinology 105: 1410, 1979)
and through Biofish Associates (Gloucester, MA). L-[3H]Tryptophan (5.9 Ci/mmol), L-[35S]cystine (483.5 Ci/mmol), and Aquasol were purchased from New England Nuclear Corp. (Boston, MA). Bio-Gel P-2 and P-10 (100-200 mesh) and CM Bio-Gel A were obtained from Bio-Rad Laboratories (Rockville Center, NY). Gel filtration of islet extracts Islets were decapsulated and 1-5 g were homogenized in a Potter-Elvejhem homogenizer in 5-10 ml 2 M acetic acid at 4 C. The homogenate was allowed to stand at 4 C overnight. The insoluble residue was removed by centrifugation and reextracted three times with 2 M acetic acid. Supernates were pooled and 5-ml samples were subjected to gel filtration on 2.5 X 20cm columns of Bio-Gel P-2 in 2 M acetic acid to remove small peptides, free amino acids, and ionic components from the crude extract. Void volume pools were collected from the P-2 columns, lyophilized, and resuspended in 2.5 ml 2 M acetic acid for filtration (20-40 mg material/column) on 1.6 X 94-cm columns of Bio-Gel P-10 to collect the crude SRIF fraction, as previously described (6, 7). Determination of protein content in extracts and preparations derived during purification was made using the Bio-Rad protein assay or by amino acid analysis (see below). Ion exchange chromatography The crude SRIF preparation recovered from P-10 columns was lyophilized and 5-10 mg of this material were suspended in 20-30 ml starting buffer (0.02 M glycine, 0.1 M urea, and 0.02% NaN3, pH 7.5). These samples were pumped at 25 ml/h onto 0.9 x 20-cm columns of CM Bio-Gel A equilibrated in starting buffer. Eluate conductivity was monitored by a Serfass conductivity bridge (Industrial Instruments Inc., Cedar Grove, NJ).
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ANGLERFISH ISLET SRIF STRUCTURE The conductivity of the starting buffer was 0.35 mmho, and SRIF-containing samples were diluted with sufficient amounts of starting buffer to bring the conductivity of each sample to 0.65-0.70 mmho. Material bound to the columns was displaced by introducing a linear salt gradient formed by running 100 ml starting buffer against 100 ml 0.5 M starting buffer in NaCl in a Pharmacia GM-1 gradient maker (Pharmacia Fine Chemicals, Piscataway, NJ). The SRIF-containing portions of eluates were lyophilized, desalted on Bio-Gel P-2, and then subjected to filtration on a 1.0 X 48-cm column of Bio-Gel P-4 in 2 M acetic acid. High pressure liquid chromatography (HPLC) The HPLC apparatus consisted of a model 204 liquid chromatograph equipped with two model 6000A pumps, a model 660 gradient programmer, and a model 450 variable wavelength detector from Waters Associates (Milford, MA). Eluate absorbance was recorded on a model 455 chart recorder from Linear Instruments Corp. (Irvine, CA) and peak integration was performed by a Minigrator from Spectra-Physics (Santa Clara, CA). Reverse phase HPLC was performed on 0.39 x 30-cm juBondapak Cig columns (Waters Associates) using 0.25 N triethylammonium formate, pH 3.0, or 0.25 N triethylammonium phosphate, pH 3.0, containing 24-26% (vol/vol) acetonitrile, as previously described (8). Samples were run at ambient temperature with a flow rate of 1.5 ml/min and a column pressure of 1500 psi. RIAs Extracts, column eluates, or isolated products were monitored for SRIF immunoreactivity by four separate SRIF RIAs. The assay systems employed were those of Patel and Reichlin using the centrally directed antiserum R149 (9,10), that of Elde using the centrally directed antiserum R141 (7,11), that of Vale et al. (9) using the N-terminally directed S39 antiserum, and that of Vale et al. (12) using the centrally directed S201.
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Instruments, Palo Alto, CA) equipped with a model 126 data system. A single column sodium citrate program (Beckman) employing Beckman A A-10 resin in a 0.28 X 20-cm column with ninhydrin detection was used. The recorder output and integrator computations from the analyzer were used to calculate amino acid and protein concentrations. Sequence determination Purified AF SRIF was carboxymethylated as previously described (5). The sequential Edman degradation of carboxymethylated AF SRIF was performed in a Beckman 890C automated spinning cup sequencer using a modification (5) of the program of Hunkapillar and Hood (15). The identity of the resulting phenylthiohydantoin (PTH) amino acids was determined by thin layer chromatography (TLC) (16) and amino acid analysis after back hydrolysis (17). The identities of PTHSer, PTH-Trp, and PTH-Asn were confirmed by TLC (16). Threonine was identified in hydrolysates of PTH amino acids as a-aminobutyric acid in the amino acid analyzer chromatogram (17).
Results Ion exchange chromatography The crude (gel filtration) preparations of AF SRIF were subjected to ion exchange chromatography on CM Bio-Gel A at pH 7.5. The results from a representative ion exchange run are shown in Fig. 1. For this experiment, unlabeled starting material (300 jug protein) was supplemented with ~50 /ig of a fraction from islet extract containing 3H-35S doubly labeled AF SRIF. The labeled islet SRIF was prepared by incubating islet tissue for 6 h with [3H]TRP and [35S]CYS (6) and then separating i 80
340
Bioassay Purified AF SRIF was tested for the ability to inhibit the release of GH from rat anterior pituitary cells maintained in culture using the method described by Vale et al. (13). Four days after plating at 5 X 105 cells/dish, the primary cultures were incubated for 3 h in 1.5 ml Hepes-modified Dulbecco's modified Eagle's medium and 2% fetal calf serum with variable doses of synthetic tetradecapeptide and AF SRIF. IBMX1 (0.4 HIM) was added to stimulate GH release. Accumulation of GH in the culture medium was monitored by GH RIA (13). Amino acid analysis Purified AF SRIF (0.5-3 jtig) was hydrolyzed in evacuated tubes with 4 N methane sulfonic acid and 0.2% tryptamine for 24 h at 110 C, as described by Moore (14). After neutralization by adding an equal volume of 3.5 N NaOH, amino acid analyses were performed using a Beckman 121 MB analyzer (Beckman 1
The following abbreviation is used: IBMX, 3-isobutyl-l-methylxanthine.
10
40 70 Fraction Number
FIG. 1. Ion exchange chromatography of AF SRIF. The SRIF-containing portions of gel filtration eluates (see Materials and Methods) were lyophilized, and the resulting material was applied in amounts of 2-10 mg to 0.9 x 20-cm columns of CM Bio-Gel A in 0.02 M glycine, 0.1 M urea, 0.02% NaN3, pH 7.5. A linear salt gradient was formed by running 100 ml starting buffer against 0.5 M 100 ml starting buffer in NaCl. In the run shown here, [3H]TRP-[35S]CYS doubly labeled SRIF purified by gel filtration (6) was added as a marker. Ninety-five percent of the SRIF immunoreactivity recovered coeluted with a 3H-35S doubly labeled moiety. The RIA was performed using the Elde R141 antiserum. • — • , disintegrations per min [3H]TRP; O--O, disintegrations per min [35S]CYS; A—A, micrograms of IRS; D - D , millimho.
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NOE ET AL.
the SRIF-sized peptide(s) from other components in the acetic acid extract by gel filtration. In addition to conductivity, radioactivity, and SRIF immunoreactivity (shown in Fig. 1), optical absorbance of the eluate was monitored at 280 nm. All such column runs generated three UV absorbance peaks. The first, representing 79% of the total absorbance, eluted before initiation of the salt gradient and had an elution volume which corresponded with the 35S radioactivity maximum in the elution profile (Fig. 1). The second UV peak eluted at 3.7 mmho and represented 12% of the total absorbance. The third absorbance peak coeluted with the elution volume of 95% of the recovered immunoreactive SRIF (IRS) and the 3H-35S doubly labeled material at 7.6 millimho. This peak represented 9% of the total monitored absorbance. Estimates of the proportion of the total absorbance contributed by each peak are based on averages from four separate carboxymethyl Bio-Gel column runs. On the basis of recovery of IRS and the presence of the expected 3 H to 35S ratio (6), this third peak was considered to be a crude preparation of AF SRIF. After desalting on BioGel P-2 and gel filtration of this material on Bio-Gel P-4, final purification of AF SS was achieved by HPLC. HPLC After having been partially purified by carboxymethyl ion exchange chromatography, the AF SRIF was subjected to HPLC, as described in Materials and Methods. The results from a typical HPLC run on a sample which contained [3H]TRP-labeled AF SRIF are shown in Fig. 2. It can be seen that the major UV absorbance peak and [3H]TRP-labeled peak eluted with the same retention time (RT). The difference of approximately 50 sec between RTs of synthetic SRIF and AF SRIF is not significant. It can be nearly completely accounted for by the difference in sample volumes applied in the two runs. Moreover, when synthetic and AF SRIF were subjected to HPLC under identical conditions after injection in the same sample volume, the difference in RT was only 2.3 sec (data not shown). Thus, AF SRIF has essentially the same RT as synthetic SRIF on HPLC. These results suggest that AF SRIF is similar to mammalian hypothalamic (synthetic) SRIF, since most synthetic SRIF analogs analyzed by reverse phase HPLC under similar conditions could be separated or at least were different enough to show a shoulder on or broadening of the SRIF peak when run as a mixture (18). One preparation of AF SRIF, which was obtained from tissue which had partially thawed because of a shipping delay, contained significant amounts of a product having a RT 1.4 min less than that of AF SRIF (RT of AF SRIF with the conditions employed, 19.2 min). This component was isolated and samples were subjected to amino acid analysis. Results from these amino acid analyses were
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8-
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25
FIG. 2. Reverse phase HPLC of synthetic and partially purified AF SRIF. Samples were subjected to isocratic elution in 0.25 N triethylammonium formate, pH 3.0, and 25.8% (vol/vol) acetonitrile. Twenty micrograms of synthetic SRIF was applied in a 40-jul sample volume. The RT of SRIF under these conditions was 14.9 min (•••). AF SRIF labeled with [ 'H]TRP was subjected to carboxymethylose ion exchange chromatography, as in Fig. 1. After desalting the appropriate pool, a 1ml sample containing approximately 50 jug protein was subjected to HPLC ( ). The major peak had a RT of 15.7 min. H, Size of the fractions collected (width) and 'H-labeled radioactivity recovered from each fraction (height). Eluate absorbance was monitored at 280 nm with a full scale setting of 0.02.
consistent with the postulate that this peptide was Des[Ala'-Gly^SRIF (Table 1, column 2). Furthermore, its RT on HPLC was identical with that of synthetic Des[Ala'-Gly'jSRIF. If this peptide is des-[Ala'-Gly2]SRIF, then the existence of an aminopeptidase in the anglerfish islet is suggested. Chemical modification of AF SS AF SRIF was modified by carboxymethylation as previously described (5). With this procedure, dicarboxymethylcysteine-SRIF (CM-SRIF) was formed. When subjected to reverse phase HPLC (Fig. 3), carboxymethylated AF SRIF and carboxymethylated hypothalamic SRIF had the same RTs. A mixture of CM-AF SRIF and CM-SRIF migrated together and formed a symmetric peak without any shoulder, indicating a high degree of similarity between CM-AF SRIF and hypothalamic CMSRIF (Fig. 3).
RIAs In previous work, it was found that AF SRIF is readily detected by immunohistochemical means or in RIAs employing antisera generated in response to injected synthetic SRIF (6, 7, 1.9). As part of the present study,
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ANGLERFISH ISLET SRIF STRUCTURE TABLE 1. Amino acid composition of AF SRIF SRIF deriva-
Amino acid
AF SRIF Hypothative (mol amino lamic and (mol amino acidVmol ala- pancreatic acid"/mol SRIF C nine) phenylalanine)
Asx Thr Ser
2.2 (2) 0.9 (1)
Glx Pro Gly Ala
0.0 0.0 0.2 0.0
'/*Cys
1.6 (2)
Val
0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0
3.0 (3) 1.9 (2)
3.0 (3) 1.9 (2)
Met He
Leu Tyr Phe Lys His Trp Arg
l.KD
1.0(1) 2.0 (2) 0.9 (1)
1 2 1
0.0 0.0
1.0(1) 1.0(1) 1.8 (2)
1 1 2
0.0 0.0
0.0
0.0
1-0 (1)
0.9 (1)
0.0
0.0 12
14
3 2 1
IT
" Samples were hydrolyzed 24 h at 110 C in 4 N methane sulfonic acid and 0.2% tryptamine. Data shown are averages from two separate analyses. * Hydrolysis was as stated above. The amount analyzed was approximately 150 pmol/amino acid. The SD from three analyses was ±0.02 mol/mol alanine. Values for serine and threonine are corrected for losses during hydrolysis. c Theoretical values, see Refs. 2-5.
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the immunoreactivities of purified AF SRIF were compared in immunoassays employing the S39 and S201 antisera. The S39 serum contains antibodies which are N-terminus specific (9) and the S201 serum is centrally directed (12). Synthetic SRIF shows the same activity in the two assays, whereas most SRIF analogs as well as larger forms of SRIF produce dissimilar immunological activities when tested with these two antisera (12). AF SRIF produced an S39 to S201 activity ratio of 1.02 ± 0.07 (SD), indicating close similarity to synthetic SRIF. Bioassay As shown in Fig. 4, AF SRIF inhibited GH release from cultured rat anterior pituitary cells in parallel with synthetic tetradecapeptide SRIF. The data were tested using the Harvard University bioassay computer program. The relative biological potency of AF SRIF compared to synthetic SRIF was calculated to be 103%, with 95% fiducial limits of 64% and 166%. These results indicate that there is no significant difference between the biological potency of AF SRIF and that of synthetic SRIF. Determination of amino acid composition The final yield of AF SRIF after carboxymethyl ion exchange chromatography was 25.5 jug AF SRIF/g islet tissue. Samples containing 1-3 jug AF SRIF purified by HPLC were subjected to amino acid analysis after hy-
.02 r
10
T CONTROL
LJ
a:
6
O
x X r-
o
ro \ 4
AF
a: \
CD a> c
10
15 TIME (mm)
20
25
FIG. 3. Reverse phase HPLC of carboxymethylated synthetic SRIF and carboxymethylated AF SRIF. Samples were subjected to isocratic elution in 0.25 N triethylammonium phosphate, pH 3.0, and 24.6% (vol/ vol) acetonitrile. • • -, Elution pattern of ~0.2 jug CM-AF SRIF alone under these conditions. , Elution pattern of 0.2 ng CM-AF SRIF and 75 ng CM-SRIF combined and injected together. The elution position of CM-AF SRIF is the same as that of CM-SRIF, with a RT of 17.9 min. Eluate absorbance was monitored at 210 nm with a full scale setting of 0.02.
0L
0 0.18
0.54
1.8
5.4
18
ng PEPTIDE FIG. 4. Effects of synthetic and AF SRIF on GH release from cultured rat anterior pituitary cells. Amounts of purified AF SRIF added are expressed as SRIF equivalents, as determined by RIA for SRIF. The points on the dose-response curve are the means ± SEM of the GH levels in three dishes. SS, Synthetic SRIF; AF SS, anglerfish somatostatin.
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NOE ET AL.
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drolysis. The concentrations of the amino acids found by the amino acid analyzer were normalized to alanine. As seen in Table 1, column 3, the calculated concentrations of nearly all of the amino acids approximated integer values, indicating that the original sample was homogeneous. The amino acid composition of AF SRIF was found to be identical with that of hypothalamic and pancreatic SRIF (2-5). Amino acid sequence Two separate sequence analyses of CM-AF SRIF were performed using 22.7 and 16.0 nmol starting material. Most of the PTH amino acids were determined directly by amino acid analysis of back hydrolysates (17). The differentiation of aspartic acid from asparagine (residue 5) and the identification of PTH-tryptophan (residue 8) and PTH-serine (residue 13) were achieved by TLC (16). The average repetitive yield for phenylalanine from both sequencer runs was 95.8%. The results demonstrated that the primary structure of AF SRIF is H-Ala-Gly-Cys-LysAsn-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH.
Discussion No specific efforts to inhibit proteolytic activity were made during extraction of the islet tissue, since after decapsulation, anglerfish islets are essentially devoid of contaminating exocrine tissue. Previous work has shown that when the SRIF localized in anglerfish islets by immunohistochemical means (19) is extracted under the conditions employed in the present study, the resulting crude preparations contain a component which has the molecular size, immunological properties, and electrophoretic mobility of synthetic SRIF (6, 7). In the present study, we have demonstrated that after purification of the crude gel filtration preparation by ion exchange chromatography (Fig. 1) and HPLC (Figs. 2 and 3), the isolated product has immunological (see Results) and biological (Fig. 4) properties indistinguishable from those of synthetic SRIF. Moreover, the amino acid composition (Table 1, column 3) and sequence were found to be identical with SRIF from sheep hypothalamus (3), pig hypothalamus (4), and pigeon pancreas (5). Whether AF SRIF contains an internal disulfide bridge like SRIF from other sources characterized previously was not determined directly by experimental means. However, it was observed that AF SRIF coeluted with cyclic SRIF on HPLC (Fig. 2) under conditions which resolve cyclic and linear SRIF. It is therefore concluded that AF SRIF is cyclized as well. Since in most preparations fragments of SRIF were not detected, we conclude that SRIF from anglerfish islets is somewhat resistant to degradation during isolation with the procedures employed. However, a peptide
Kudo i 1979 Vol 105 i No 6
which has the amino acid composition and HPLC retention time of des-fAla'-Gly^SRIF was found in a preparation of SRIF derived from tissue which had thawed partially during shipment (Table 1, column 2). Although this observation suggests the existence of aminopeptidase(s) in anglerfish islet tissue, no attempt to confirm the presence of specific proteases was made in the present study. Peptides having the immunological characteristics of SRIF have been localized by RIA or immunohistochemical means in the gut or pancreas of several other species of teleost fish (9, 19-21), reptiles (20, 22), the hagfish, a cyclostome (9, 20), and the protochordate tunicate Ciona intestinalis (23). Whether the peptide with SRIF-like immunoactivity found in all of these species has the same primary structure and/or biological activity as synthetic SRIF remains to be determined. As indicated by the results from the present study, AF SRIF is identical to pigeon pancreatic SRIF and ovine and porcine hypothalamic SRIF. The identification in a teleost fish of a biologically active peptide which has the same primary structure as SRIF isolated from both nonneural and neural tissues of phylogenetically more advanced species indicates a low frequency of mutation for the portion of the genome which codes for this peptide. The fact that the primary structure of tetradecapeptide SRIF from two different tissue sources has been maintained throughout phylogeny from a teleost fish to birds and mammals argues that SRIF has served as an important regulatory peptide for a very long period of time indeed.
Acknowledgments The skillful technical assistance of Carolyn Williams, Gail Debo, Carolyn Douglas, Pam Smith, and Frances Lux is gratefully acknowledged. We also wish to thank Margaret Pierce and Sue Hebert for help in preparation of this manuscript. Our appreciation to Dr. R. P. Elde, University of Minnesota, who supplied us with antiserum R141. Our thanks also to Don Flescher and Henry Jensen of the National Marine Fisheries Service (Woods Hole) who provided some of the islet tissue employed in this study.
References 1. Luft, R., S. Efendic, and T. Hokfelt, Somatostatin—both hormone and neurotransmitter? Diabetologia 14: 1, 1978. 2. Brazeau, P., W. Vale, R. Burgus, N. Ling, M. Butcher, J. Rivier, and R. Guillemin, Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone, Science 179: 77, 1973. 3. Burgus, R., N. Ling, M. Butcher, and R. Guillemin, Primary structure of somatostatin, a hypothalamic peptide that inhibits the secretion of pituitary growth hormone, Proc Natl Acad Sci USA 70: 684, 1973. 4. Schally, A. V., A. Dupont, A. Arimura, T. W. Redding, N. Nishi, G. L. Linthicum, and D. H. Schlesinger, Isolation and structure of somatostatin from porcine hypothalami, Biochemistry 15: 509, 1976. 5. Spiess, J., J. E. Rivier, J. A. Rodkey, C. D. Bennett, and W. Vale,
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ANGLERFISH ISLET SRIF STRUCTURE Isolation and characterization of somatostatin from pigeon pancreas, Proc Natl Acad Sci USA 76: 2974, 1979. 6. Noe, B. D., D. J. Fletcher, G. E. Bauer, G. C. Weir, and Y. Patel, Somatostatin biosynthesis occurs in pancreatic islets, Endocrinology 102: 1675, 1978. 7. Noe, B. D., D. J. Fletcher, and J. Spiess, Evidence for the existence of a biosynthetic precursor for somatostatin, Diabetes 28: 724,1979. 8. Rivier, J., Use of trialkyl ammonium phosphate (TAAP) buffers in reverse phase HPLC for high resolution and high recovery of peptides and proteins, J Liq Chromatogr 1: 343, 1978. 9. Vale, W., N. Ling, J. Rivier, J. Villarreal, C. Rivier, C. Douglas, and M. Brown, Anatomic and phylogenetic distribution of somatostatin, Metabolism 25: 1491, 1976. 10. Patel, Y., and S. Reichlin, Somatostatin in hypothalamus, extrahypothalamic brain, and peripheral tissues of the rat, Endocrinology 102: 523, 1978. 11. Arimura, A., G. Lundqvist, J. Rothman, R. Chang, R. FernandezDurango, R. Elde, D. H. Coy, C. Meyers, and A. V. Schally, Radioimmunoassay of somatostatin, Metabolism 27: 1139, 1978. 12. Vale, W., J. Rivier, N. Ling, and M. Brown, Biologic and immunologic activities and applications of somatostatin analogs, Metabolism 27: 1391, 1978. 13. Vale, W., P. Brazeau, C. Rivier, M. Brown, B. Boss, J. Rivier, R. Burgus, N. Ling, and R. Guillemin, Somatostatin, Recent Prog Horm Res 31: 365, 1975. 14. Moore, S., The precision and sensitivity of amino acid analysis, In Meienhofer, J. (ed.), Chemistry and Biology of Peptides, Ann Arbor Science Publishers, Ann Arbor, 1972, p. 629. 15. Hunkapillar, M. W., and L. E. Hood, Direct microsequence analysis of polypeptides using an improved sequenator, a nonprotein carrier
16.
17. 18. 19.
20. 21. 22. 23.
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(Polybrene), and high pressure liquid chromatography, Biochemistry 17: 2124, 1978. Kulbe, K. D., Micropolyamide thin layer chromatography ofphenylthiohydantoin amino acids (PTH) at subnanomolar level. A rapid microtechnique for simultaneous multisample identification after automated Edman degradation, Anal Biochem 59: 564, 1974. Mendez, E., and C. Y. Lai, Regeneration of amino acids from thiazolinones formed in the Edman degradation, Anal Biochem 68: 47, 1975. Burgus, R., and J. Rivier, Use of high pressure liquid chromatography in the purification of peptides, In Loffet, A. (ed.), Peptides 1976, Editions de l'Universite de Bruxelles, Belgium, 1976, p. 85. Johnson, D. E., J. L. Torrence, R. P. Elde, G. E. Bauer, B. D. Noe, and D. J. Fletcher, Immunohistochemical localization of somatostatin, insulin and glucagon in the principal islets of the anglerfish (Lophius americanus) and the channel catfish (Ictalurus punctatus), Am JAnat 147: 119, 1976. Falkmer, S., and Y. Ostberg, Comparative morphology of pancreatic islets in animals, In Volk, B. W., and K. F. Wellmann (eds.), The Diabetic Pancreas, Plenum Press, New York, 1976. Van Noorden, S., and G. J. Patent, Localization of pancreatic polypeptide (PP)-like immunoreactivity in the pancreatic islets of some teleost fishes, Cell Tissue Res 188: 521, 1978. Rhoten, W. B., and P. H. Smith, Localization of four polypeptide hormones in the saurian pancreas, Am J Anat 151: 595, 1978. Fritsch, H. A. R., S. Van Noorden, and A. G. E. Pearse, Localisation of somatostatin- and gastrin-like immunoreactivity in the gastrointestinal tract of Ciona intestinalis L., Cell Tissue Res 186: 181, 1978.
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Vol. 105, No. 6 Printed in U.S.A.
Isolation and Characterization of Somatostatin from Anglerfish Pancreatic Islet* BRYAN D. NOE,f JOACHIM SPIESS, JEAN E. RIVIER, AND WYLIE VALE Department of Anatomy, Emory University School of Medicine, Atlanta, Georgia 30322 and Marine Biological Laboratory, Woods Hole, Massachusetts 02543 (B.D.N.); and the Peptide Biology Laboratory at The Salk Institute for Biological Studies (J.S., J.E.R. and W. V.), La Jolla, California 92037
ABSTRACT. Somatostatin was purified from anglerfish pancreatic islets using acetic acid extraction, gel filtration (Bio-Gel P-10), ion exchange chromatography (CM Bio-Gel A), and reversed phase high pressure liquid chromatography. The resulting peptide was characterized by RIA, bioassay, and determination of amino acid composition. Anglerfish islet somatostatin was found to possess an amino acid composition and immunological
S
OMATOSTATIN (SRIF) has been localized by bioassay and/or immunological techniques in numerous tissues (1). It is not known, however, whether the SRIF-like species identified in all of these sites are identical in structure and biological activity. To date, only the primary structures of SRIF from ovine hypothalamus (2, 3), porcine hypothalamus (4), and pigeon pancreas (5) have been determined. SRIF isolated from each of these sources was found to have the same primary structure. It has been demonstrated recently that the biosynthesis of pancreatic islet SRIF involves a precursor-product pathway (6, 7). As part of an investigative effort designed to isolate and characterize the biosynthetic precursor for anglerfish islet SRIF (AF SRIF), we have purified and characterized tetradecapeptide SRIF from this source. We report here the results of this work. Materials and Methods
Materials Anglerfish islets (25 g) were obtained at the Marine Biological Laboratory (Woods Hole, MA), the Northeast Fisheries Center of the National Marine Fisheries Service (Woods Hole, MA), Received December 21, 1978. Address all correspondence and requests for reprints to: Dr. Bryan D. Noe, Department of Anatomy, Emory University School of Medicine, Atlanta, Georgia 30322. * This work was supported in part by NIH Grants AM-19890 and AM-16921 (to B.D.N.) and AM-20917 and AM-18811, National Foundation Grant 1-411, and a grant from the Texas Research Foundation/ Salk Institute (Peptide Biology Lab). f Recipient of a USPHS Research Career Development Award AM00142.
and biological activities equivalent to synthetic somatostatin. Sequence analyses revealed that the primary structure was HAla-Gly-cyclo-[Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-ThrSer-Cys]-OH. These results demonstrate that anglerfish islet somatostatin has the same primary structure as somatostatin from all other sources characterized to date. (Endocrinology 105: 1410, 1979)
and through Biofish Associates (Gloucester, MA). L-[3H]Tryptophan (5.9 Ci/mmol), L-[35S]cystine (483.5 Ci/mmol), and Aquasol were purchased from New England Nuclear Corp. (Boston, MA). Bio-Gel P-2 and P-10 (100-200 mesh) and CM Bio-Gel A were obtained from Bio-Rad Laboratories (Rockville Center, NY). Gel filtration of islet extracts Islets were decapsulated and 1-5 g were homogenized in a Potter-Elvejhem homogenizer in 5-10 ml 2 M acetic acid at 4 C. The homogenate was allowed to stand at 4 C overnight. The insoluble residue was removed by centrifugation and reextracted three times with 2 M acetic acid. Supernates were pooled and 5-ml samples were subjected to gel filtration on 2.5 X 20cm columns of Bio-Gel P-2 in 2 M acetic acid to remove small peptides, free amino acids, and ionic components from the crude extract. Void volume pools were collected from the P-2 columns, lyophilized, and resuspended in 2.5 ml 2 M acetic acid for filtration (20-40 mg material/column) on 1.6 X 94-cm columns of Bio-Gel P-10 to collect the crude SRIF fraction, as previously described (6, 7). Determination of protein content in extracts and preparations derived during purification was made using the Bio-Rad protein assay or by amino acid analysis (see below). Ion exchange chromatography The crude SRIF preparation recovered from P-10 columns was lyophilized and 5-10 mg of this material were suspended in 20-30 ml starting buffer (0.02 M glycine, 0.1 M urea, and 0.02% NaN3, pH 7.5). These samples were pumped at 25 ml/h onto 0.9 x 20-cm columns of CM Bio-Gel A equilibrated in starting buffer. Eluate conductivity was monitored by a Serfass conductivity bridge (Industrial Instruments Inc., Cedar Grove, NJ).
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ANGLERFISH ISLET SRIF STRUCTURE The conductivity of the starting buffer was 0.35 mmho, and SRIF-containing samples were diluted with sufficient amounts of starting buffer to bring the conductivity of each sample to 0.65-0.70 mmho. Material bound to the columns was displaced by introducing a linear salt gradient formed by running 100 ml starting buffer against 100 ml 0.5 M starting buffer in NaCl in a Pharmacia GM-1 gradient maker (Pharmacia Fine Chemicals, Piscataway, NJ). The SRIF-containing portions of eluates were lyophilized, desalted on Bio-Gel P-2, and then subjected to filtration on a 1.0 X 48-cm column of Bio-Gel P-4 in 2 M acetic acid. High pressure liquid chromatography (HPLC) The HPLC apparatus consisted of a model 204 liquid chromatograph equipped with two model 6000A pumps, a model 660 gradient programmer, and a model 450 variable wavelength detector from Waters Associates (Milford, MA). Eluate absorbance was recorded on a model 455 chart recorder from Linear Instruments Corp. (Irvine, CA) and peak integration was performed by a Minigrator from Spectra-Physics (Santa Clara, CA). Reverse phase HPLC was performed on 0.39 x 30-cm juBondapak Cig columns (Waters Associates) using 0.25 N triethylammonium formate, pH 3.0, or 0.25 N triethylammonium phosphate, pH 3.0, containing 24-26% (vol/vol) acetonitrile, as previously described (8). Samples were run at ambient temperature with a flow rate of 1.5 ml/min and a column pressure of 1500 psi. RIAs Extracts, column eluates, or isolated products were monitored for SRIF immunoreactivity by four separate SRIF RIAs. The assay systems employed were those of Patel and Reichlin using the centrally directed antiserum R149 (9,10), that of Elde using the centrally directed antiserum R141 (7,11), that of Vale et al. (9) using the N-terminally directed S39 antiserum, and that of Vale et al. (12) using the centrally directed S201.
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Instruments, Palo Alto, CA) equipped with a model 126 data system. A single column sodium citrate program (Beckman) employing Beckman A A-10 resin in a 0.28 X 20-cm column with ninhydrin detection was used. The recorder output and integrator computations from the analyzer were used to calculate amino acid and protein concentrations. Sequence determination Purified AF SRIF was carboxymethylated as previously described (5). The sequential Edman degradation of carboxymethylated AF SRIF was performed in a Beckman 890C automated spinning cup sequencer using a modification (5) of the program of Hunkapillar and Hood (15). The identity of the resulting phenylthiohydantoin (PTH) amino acids was determined by thin layer chromatography (TLC) (16) and amino acid analysis after back hydrolysis (17). The identities of PTHSer, PTH-Trp, and PTH-Asn were confirmed by TLC (16). Threonine was identified in hydrolysates of PTH amino acids as a-aminobutyric acid in the amino acid analyzer chromatogram (17).
Results Ion exchange chromatography The crude (gel filtration) preparations of AF SRIF were subjected to ion exchange chromatography on CM Bio-Gel A at pH 7.5. The results from a representative ion exchange run are shown in Fig. 1. For this experiment, unlabeled starting material (300 jug protein) was supplemented with ~50 /ig of a fraction from islet extract containing 3H-35S doubly labeled AF SRIF. The labeled islet SRIF was prepared by incubating islet tissue for 6 h with [3H]TRP and [35S]CYS (6) and then separating i 80
340
Bioassay Purified AF SRIF was tested for the ability to inhibit the release of GH from rat anterior pituitary cells maintained in culture using the method described by Vale et al. (13). Four days after plating at 5 X 105 cells/dish, the primary cultures were incubated for 3 h in 1.5 ml Hepes-modified Dulbecco's modified Eagle's medium and 2% fetal calf serum with variable doses of synthetic tetradecapeptide and AF SRIF. IBMX1 (0.4 HIM) was added to stimulate GH release. Accumulation of GH in the culture medium was monitored by GH RIA (13). Amino acid analysis Purified AF SRIF (0.5-3 jtig) was hydrolyzed in evacuated tubes with 4 N methane sulfonic acid and 0.2% tryptamine for 24 h at 110 C, as described by Moore (14). After neutralization by adding an equal volume of 3.5 N NaOH, amino acid analyses were performed using a Beckman 121 MB analyzer (Beckman 1
The following abbreviation is used: IBMX, 3-isobutyl-l-methylxanthine.
10
40 70 Fraction Number
FIG. 1. Ion exchange chromatography of AF SRIF. The SRIF-containing portions of gel filtration eluates (see Materials and Methods) were lyophilized, and the resulting material was applied in amounts of 2-10 mg to 0.9 x 20-cm columns of CM Bio-Gel A in 0.02 M glycine, 0.1 M urea, 0.02% NaN3, pH 7.5. A linear salt gradient was formed by running 100 ml starting buffer against 0.5 M 100 ml starting buffer in NaCl. In the run shown here, [3H]TRP-[35S]CYS doubly labeled SRIF purified by gel filtration (6) was added as a marker. Ninety-five percent of the SRIF immunoreactivity recovered coeluted with a 3H-35S doubly labeled moiety. The RIA was performed using the Elde R141 antiserum. • — • , disintegrations per min [3H]TRP; O--O, disintegrations per min [35S]CYS; A—A, micrograms of IRS; D - D , millimho.
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NOE ET AL.
the SRIF-sized peptide(s) from other components in the acetic acid extract by gel filtration. In addition to conductivity, radioactivity, and SRIF immunoreactivity (shown in Fig. 1), optical absorbance of the eluate was monitored at 280 nm. All such column runs generated three UV absorbance peaks. The first, representing 79% of the total absorbance, eluted before initiation of the salt gradient and had an elution volume which corresponded with the 35S radioactivity maximum in the elution profile (Fig. 1). The second UV peak eluted at 3.7 mmho and represented 12% of the total absorbance. The third absorbance peak coeluted with the elution volume of 95% of the recovered immunoreactive SRIF (IRS) and the 3H-35S doubly labeled material at 7.6 millimho. This peak represented 9% of the total monitored absorbance. Estimates of the proportion of the total absorbance contributed by each peak are based on averages from four separate carboxymethyl Bio-Gel column runs. On the basis of recovery of IRS and the presence of the expected 3 H to 35S ratio (6), this third peak was considered to be a crude preparation of AF SRIF. After desalting on BioGel P-2 and gel filtration of this material on Bio-Gel P-4, final purification of AF SS was achieved by HPLC. HPLC After having been partially purified by carboxymethyl ion exchange chromatography, the AF SRIF was subjected to HPLC, as described in Materials and Methods. The results from a typical HPLC run on a sample which contained [3H]TRP-labeled AF SRIF are shown in Fig. 2. It can be seen that the major UV absorbance peak and [3H]TRP-labeled peak eluted with the same retention time (RT). The difference of approximately 50 sec between RTs of synthetic SRIF and AF SRIF is not significant. It can be nearly completely accounted for by the difference in sample volumes applied in the two runs. Moreover, when synthetic and AF SRIF were subjected to HPLC under identical conditions after injection in the same sample volume, the difference in RT was only 2.3 sec (data not shown). Thus, AF SRIF has essentially the same RT as synthetic SRIF on HPLC. These results suggest that AF SRIF is similar to mammalian hypothalamic (synthetic) SRIF, since most synthetic SRIF analogs analyzed by reverse phase HPLC under similar conditions could be separated or at least were different enough to show a shoulder on or broadening of the SRIF peak when run as a mixture (18). One preparation of AF SRIF, which was obtained from tissue which had partially thawed because of a shipping delay, contained significant amounts of a product having a RT 1.4 min less than that of AF SRIF (RT of AF SRIF with the conditions employed, 19.2 min). This component was isolated and samples were subjected to amino acid analysis. Results from these amino acid analyses were
Kndo Vol 105
1979 No 6
1.02
8-
10
15 20 Time (Min)
25
FIG. 2. Reverse phase HPLC of synthetic and partially purified AF SRIF. Samples were subjected to isocratic elution in 0.25 N triethylammonium formate, pH 3.0, and 25.8% (vol/vol) acetonitrile. Twenty micrograms of synthetic SRIF was applied in a 40-jul sample volume. The RT of SRIF under these conditions was 14.9 min (•••). AF SRIF labeled with [ 'H]TRP was subjected to carboxymethylose ion exchange chromatography, as in Fig. 1. After desalting the appropriate pool, a 1ml sample containing approximately 50 jug protein was subjected to HPLC ( ). The major peak had a RT of 15.7 min. H, Size of the fractions collected (width) and 'H-labeled radioactivity recovered from each fraction (height). Eluate absorbance was monitored at 280 nm with a full scale setting of 0.02.
consistent with the postulate that this peptide was Des[Ala'-Gly^SRIF (Table 1, column 2). Furthermore, its RT on HPLC was identical with that of synthetic Des[Ala'-Gly'jSRIF. If this peptide is des-[Ala'-Gly2]SRIF, then the existence of an aminopeptidase in the anglerfish islet is suggested. Chemical modification of AF SS AF SRIF was modified by carboxymethylation as previously described (5). With this procedure, dicarboxymethylcysteine-SRIF (CM-SRIF) was formed. When subjected to reverse phase HPLC (Fig. 3), carboxymethylated AF SRIF and carboxymethylated hypothalamic SRIF had the same RTs. A mixture of CM-AF SRIF and CM-SRIF migrated together and formed a symmetric peak without any shoulder, indicating a high degree of similarity between CM-AF SRIF and hypothalamic CMSRIF (Fig. 3).
RIAs In previous work, it was found that AF SRIF is readily detected by immunohistochemical means or in RIAs employing antisera generated in response to injected synthetic SRIF (6, 7, 1.9). As part of the present study,
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ANGLERFISH ISLET SRIF STRUCTURE TABLE 1. Amino acid composition of AF SRIF SRIF deriva-
Amino acid
AF SRIF Hypothative (mol amino lamic and (mol amino acidVmol ala- pancreatic acid"/mol SRIF C nine) phenylalanine)
Asx Thr Ser
2.2 (2) 0.9 (1)
Glx Pro Gly Ala
0.0 0.0 0.2 0.0
'/*Cys
1.6 (2)
Val
0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0
3.0 (3) 1.9 (2)
3.0 (3) 1.9 (2)
Met He
Leu Tyr Phe Lys His Trp Arg
l.KD
1.0(1) 2.0 (2) 0.9 (1)
1 2 1
0.0 0.0
1.0(1) 1.0(1) 1.8 (2)
1 1 2
0.0 0.0
0.0
0.0
1-0 (1)
0.9 (1)
0.0
0.0 12
14
3 2 1
IT
" Samples were hydrolyzed 24 h at 110 C in 4 N methane sulfonic acid and 0.2% tryptamine. Data shown are averages from two separate analyses. * Hydrolysis was as stated above. The amount analyzed was approximately 150 pmol/amino acid. The SD from three analyses was ±0.02 mol/mol alanine. Values for serine and threonine are corrected for losses during hydrolysis. c Theoretical values, see Refs. 2-5.
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the immunoreactivities of purified AF SRIF were compared in immunoassays employing the S39 and S201 antisera. The S39 serum contains antibodies which are N-terminus specific (9) and the S201 serum is centrally directed (12). Synthetic SRIF shows the same activity in the two assays, whereas most SRIF analogs as well as larger forms of SRIF produce dissimilar immunological activities when tested with these two antisera (12). AF SRIF produced an S39 to S201 activity ratio of 1.02 ± 0.07 (SD), indicating close similarity to synthetic SRIF. Bioassay As shown in Fig. 4, AF SRIF inhibited GH release from cultured rat anterior pituitary cells in parallel with synthetic tetradecapeptide SRIF. The data were tested using the Harvard University bioassay computer program. The relative biological potency of AF SRIF compared to synthetic SRIF was calculated to be 103%, with 95% fiducial limits of 64% and 166%. These results indicate that there is no significant difference between the biological potency of AF SRIF and that of synthetic SRIF. Determination of amino acid composition The final yield of AF SRIF after carboxymethyl ion exchange chromatography was 25.5 jug AF SRIF/g islet tissue. Samples containing 1-3 jug AF SRIF purified by HPLC were subjected to amino acid analysis after hy-
.02 r
10
T CONTROL
LJ
a:
6
O
x X r-
o
ro \ 4
AF
a: \
CD a> c
10
15 TIME (mm)
20
25
FIG. 3. Reverse phase HPLC of carboxymethylated synthetic SRIF and carboxymethylated AF SRIF. Samples were subjected to isocratic elution in 0.25 N triethylammonium phosphate, pH 3.0, and 24.6% (vol/ vol) acetonitrile. • • -, Elution pattern of ~0.2 jug CM-AF SRIF alone under these conditions. , Elution pattern of 0.2 ng CM-AF SRIF and 75 ng CM-SRIF combined and injected together. The elution position of CM-AF SRIF is the same as that of CM-SRIF, with a RT of 17.9 min. Eluate absorbance was monitored at 210 nm with a full scale setting of 0.02.
0L
0 0.18
0.54
1.8
5.4
18
ng PEPTIDE FIG. 4. Effects of synthetic and AF SRIF on GH release from cultured rat anterior pituitary cells. Amounts of purified AF SRIF added are expressed as SRIF equivalents, as determined by RIA for SRIF. The points on the dose-response curve are the means ± SEM of the GH levels in three dishes. SS, Synthetic SRIF; AF SS, anglerfish somatostatin.
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NOE ET AL.
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drolysis. The concentrations of the amino acids found by the amino acid analyzer were normalized to alanine. As seen in Table 1, column 3, the calculated concentrations of nearly all of the amino acids approximated integer values, indicating that the original sample was homogeneous. The amino acid composition of AF SRIF was found to be identical with that of hypothalamic and pancreatic SRIF (2-5). Amino acid sequence Two separate sequence analyses of CM-AF SRIF were performed using 22.7 and 16.0 nmol starting material. Most of the PTH amino acids were determined directly by amino acid analysis of back hydrolysates (17). The differentiation of aspartic acid from asparagine (residue 5) and the identification of PTH-tryptophan (residue 8) and PTH-serine (residue 13) were achieved by TLC (16). The average repetitive yield for phenylalanine from both sequencer runs was 95.8%. The results demonstrated that the primary structure of AF SRIF is H-Ala-Gly-Cys-LysAsn-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH.
Discussion No specific efforts to inhibit proteolytic activity were made during extraction of the islet tissue, since after decapsulation, anglerfish islets are essentially devoid of contaminating exocrine tissue. Previous work has shown that when the SRIF localized in anglerfish islets by immunohistochemical means (19) is extracted under the conditions employed in the present study, the resulting crude preparations contain a component which has the molecular size, immunological properties, and electrophoretic mobility of synthetic SRIF (6, 7). In the present study, we have demonstrated that after purification of the crude gel filtration preparation by ion exchange chromatography (Fig. 1) and HPLC (Figs. 2 and 3), the isolated product has immunological (see Results) and biological (Fig. 4) properties indistinguishable from those of synthetic SRIF. Moreover, the amino acid composition (Table 1, column 3) and sequence were found to be identical with SRIF from sheep hypothalamus (3), pig hypothalamus (4), and pigeon pancreas (5). Whether AF SRIF contains an internal disulfide bridge like SRIF from other sources characterized previously was not determined directly by experimental means. However, it was observed that AF SRIF coeluted with cyclic SRIF on HPLC (Fig. 2) under conditions which resolve cyclic and linear SRIF. It is therefore concluded that AF SRIF is cyclized as well. Since in most preparations fragments of SRIF were not detected, we conclude that SRIF from anglerfish islets is somewhat resistant to degradation during isolation with the procedures employed. However, a peptide
Kudo i 1979 Vol 105 i No 6
which has the amino acid composition and HPLC retention time of des-fAla'-Gly^SRIF was found in a preparation of SRIF derived from tissue which had thawed partially during shipment (Table 1, column 2). Although this observation suggests the existence of aminopeptidase(s) in anglerfish islet tissue, no attempt to confirm the presence of specific proteases was made in the present study. Peptides having the immunological characteristics of SRIF have been localized by RIA or immunohistochemical means in the gut or pancreas of several other species of teleost fish (9, 19-21), reptiles (20, 22), the hagfish, a cyclostome (9, 20), and the protochordate tunicate Ciona intestinalis (23). Whether the peptide with SRIF-like immunoactivity found in all of these species has the same primary structure and/or biological activity as synthetic SRIF remains to be determined. As indicated by the results from the present study, AF SRIF is identical to pigeon pancreatic SRIF and ovine and porcine hypothalamic SRIF. The identification in a teleost fish of a biologically active peptide which has the same primary structure as SRIF isolated from both nonneural and neural tissues of phylogenetically more advanced species indicates a low frequency of mutation for the portion of the genome which codes for this peptide. The fact that the primary structure of tetradecapeptide SRIF from two different tissue sources has been maintained throughout phylogeny from a teleost fish to birds and mammals argues that SRIF has served as an important regulatory peptide for a very long period of time indeed.
Acknowledgments The skillful technical assistance of Carolyn Williams, Gail Debo, Carolyn Douglas, Pam Smith, and Frances Lux is gratefully acknowledged. We also wish to thank Margaret Pierce and Sue Hebert for help in preparation of this manuscript. Our appreciation to Dr. R. P. Elde, University of Minnesota, who supplied us with antiserum R141. Our thanks also to Don Flescher and Henry Jensen of the National Marine Fisheries Service (Woods Hole) who provided some of the islet tissue employed in this study.
References 1. Luft, R., S. Efendic, and T. Hokfelt, Somatostatin—both hormone and neurotransmitter? Diabetologia 14: 1, 1978. 2. Brazeau, P., W. Vale, R. Burgus, N. Ling, M. Butcher, J. Rivier, and R. Guillemin, Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone, Science 179: 77, 1973. 3. Burgus, R., N. Ling, M. Butcher, and R. Guillemin, Primary structure of somatostatin, a hypothalamic peptide that inhibits the secretion of pituitary growth hormone, Proc Natl Acad Sci USA 70: 684, 1973. 4. Schally, A. V., A. Dupont, A. Arimura, T. W. Redding, N. Nishi, G. L. Linthicum, and D. H. Schlesinger, Isolation and structure of somatostatin from porcine hypothalami, Biochemistry 15: 509, 1976. 5. Spiess, J., J. E. Rivier, J. A. Rodkey, C. D. Bennett, and W. Vale,
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ANGLERFISH ISLET SRIF STRUCTURE Isolation and characterization of somatostatin from pigeon pancreas, Proc Natl Acad Sci USA 76: 2974, 1979. 6. Noe, B. D., D. J. Fletcher, G. E. Bauer, G. C. Weir, and Y. Patel, Somatostatin biosynthesis occurs in pancreatic islets, Endocrinology 102: 1675, 1978. 7. Noe, B. D., D. J. Fletcher, and J. Spiess, Evidence for the existence of a biosynthetic precursor for somatostatin, Diabetes 28: 724,1979. 8. Rivier, J., Use of trialkyl ammonium phosphate (TAAP) buffers in reverse phase HPLC for high resolution and high recovery of peptides and proteins, J Liq Chromatogr 1: 343, 1978. 9. Vale, W., N. Ling, J. Rivier, J. Villarreal, C. Rivier, C. Douglas, and M. Brown, Anatomic and phylogenetic distribution of somatostatin, Metabolism 25: 1491, 1976. 10. Patel, Y., and S. Reichlin, Somatostatin in hypothalamus, extrahypothalamic brain, and peripheral tissues of the rat, Endocrinology 102: 523, 1978. 11. Arimura, A., G. Lundqvist, J. Rothman, R. Chang, R. FernandezDurango, R. Elde, D. H. Coy, C. Meyers, and A. V. Schally, Radioimmunoassay of somatostatin, Metabolism 27: 1139, 1978. 12. Vale, W., J. Rivier, N. Ling, and M. Brown, Biologic and immunologic activities and applications of somatostatin analogs, Metabolism 27: 1391, 1978. 13. Vale, W., P. Brazeau, C. Rivier, M. Brown, B. Boss, J. Rivier, R. Burgus, N. Ling, and R. Guillemin, Somatostatin, Recent Prog Horm Res 31: 365, 1975. 14. Moore, S., The precision and sensitivity of amino acid analysis, In Meienhofer, J. (ed.), Chemistry and Biology of Peptides, Ann Arbor Science Publishers, Ann Arbor, 1972, p. 629. 15. Hunkapillar, M. W., and L. E. Hood, Direct microsequence analysis of polypeptides using an improved sequenator, a nonprotein carrier
16.
17. 18. 19.
20. 21. 22. 23.
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(Polybrene), and high pressure liquid chromatography, Biochemistry 17: 2124, 1978. Kulbe, K. D., Micropolyamide thin layer chromatography ofphenylthiohydantoin amino acids (PTH) at subnanomolar level. A rapid microtechnique for simultaneous multisample identification after automated Edman degradation, Anal Biochem 59: 564, 1974. Mendez, E., and C. Y. Lai, Regeneration of amino acids from thiazolinones formed in the Edman degradation, Anal Biochem 68: 47, 1975. Burgus, R., and J. Rivier, Use of high pressure liquid chromatography in the purification of peptides, In Loffet, A. (ed.), Peptides 1976, Editions de l'Universite de Bruxelles, Belgium, 1976, p. 85. Johnson, D. E., J. L. Torrence, R. P. Elde, G. E. Bauer, B. D. Noe, and D. J. Fletcher, Immunohistochemical localization of somatostatin, insulin and glucagon in the principal islets of the anglerfish (Lophius americanus) and the channel catfish (Ictalurus punctatus), Am JAnat 147: 119, 1976. Falkmer, S., and Y. Ostberg, Comparative morphology of pancreatic islets in animals, In Volk, B. W., and K. F. Wellmann (eds.), The Diabetic Pancreas, Plenum Press, New York, 1976. Van Noorden, S., and G. J. Patent, Localization of pancreatic polypeptide (PP)-like immunoreactivity in the pancreatic islets of some teleost fishes, Cell Tissue Res 188: 521, 1978. Rhoten, W. B., and P. H. Smith, Localization of four polypeptide hormones in the saurian pancreas, Am J Anat 151: 595, 1978. Fritsch, H. A. R., S. Van Noorden, and A. G. E. Pearse, Localisation of somatostatin- and gastrin-like immunoreactivity in the gastrointestinal tract of Ciona intestinalis L., Cell Tissue Res 186: 181, 1978.
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