BIOCHEMICAL
Vol. 169, No. 2, 1990 June 15, 1990
REGIONAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 788-795
DISTRIBUTION AND MOLECULAR FORMS OF RAT ISLET AMYLOID POLYPEPTIDE
Junko ASAIl, MasamitsuNAKAZATO’O, Mikiya MIYAZATO’, Kenji KANGAWA2, Hisayuki MATSU03 and Shigeru MATSUKURA’ Departmentsof ‘Internal Medicine and ‘Biochemistry, Miyazaki Medical College, Kiyotake, Miyazaki 889- 16, Japan 3National CardiovascularCenter ResearchInstitute, Suita, Osaka565, Japan Received
May 7, 1990
SUMMARY: Using a highly sensitive and specific radioimmunoassay(RIA) for rat islet amyloid polypeptide (IAPP), we clarified regional distribution and molecular forms of rat IAPP. IAPP[l- 371 and IAPP[19- 371were identified in normal rat pancreasby sequenceanalyses. IAPP[19- 371, accounting for 57% of IAPP- immunoreactivity in rat pancreas,is a major molecular form of rat IAPP moiety. In human,however, IAPP[l- 371 is the major component, with IAPP[17- 371composingas little as 2- 6% of IAPP- immunoreactivity in pancreas.This indicatesthat processingof IAPP in pancreasdiffers in species.A large amount of IAPP (328.5 f 25.0 pmollg wet weight) was found in rat pancreasand the peptide was also detected in pyloric antrum of the stomach,duodenum,jejunum, ileum, and colon at O.l- 0.8% of the level of pancreas. It was not detected in central nervoussystem. The content of rat IAPP in pancreas fell to 54% of control after 4 day fasting. The distribution of IAPP suggestsits possible endocrineor paracrinefunction in pancreasand gastrointestinaltract. 01990 Academic Press, I~C.
Islet amyloid polypeptide (IAPP) (l), also designatedamylin (2) is a 37- amino acid polypeptide which constitutesislet amyloid in pancreasof patients with non-insulin- dependent diabetesmellitus. IAPP is a member of the calcitonin gene- related peptide (CGRP) family. Amino acid sequencesof the two peptides are 46% identical. We have recently isolated rat IAPP from normal rat pancreasby utilizing the cross- reactivity of an antiserumraisedagainst human IAPP with rat IAPP (3). Both rat and human IAPPs are 37 amino acids long and their sequencesare 84% identical. In the present study, we establisheda sensitive and specific radioimmunoassay(RIA) system for rat IAPP basedon its sequence.Using RIA, we studied regional distribution of rat IAPP to delineate its physiological function. Furthermore, we identified IAPP[l - 371 and §To whom correspondenceshouldbe addressed. ABBREVIATIONS: CGRP, calcitonin gene- related peptide. HPLC, high performance liquid chromatography. w, islet amyloid polypeptide. k, immunoreactive. m, radioimmunoassay. 0006-291X/90 Copyright All rights
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
788
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169,
IAPP[19-
No.
2, 1990
BIOCHEMICAL
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BIOPHYSICAL
RESEARCH
COMMUNICATIONS
371 in rat pancreas by sequence analyses. Changes in the content and molecular forms
of rat IAPP in pancreas in response to fasting were also demonstrated.
MATERIALS
AND METHODS
Peptide synthesis A pentadecapeptide, termed rat IAPP-15 which corresponds to subsequence [24- 371 of rat IAPP with an additional arginine at the N- terminus, was synthesized by solid phase techniques, using peptide synthesizer Model 430A (Applied Biosystems). The peptide was purified by reverse- phase high performance liquid chromatography (HPLC) on a p - Bondasphere 5 fi C18 300A column. Correct synthesis was confirmed by amino acid analysis and sequencing. Preparation of antiserum Rat IAPP -15 (12 mg) was conjugated with bovine thyroglobulin (16 mg) by the glutaraldehyde method. The reaction mixture was dialyzed four times against one liter of 0.9% NaCl. The antigenic conjugate solution (1.5- 3 ml) was emulsified with an equal volume of Freund’s complete adjuvant, and used for immunizing New Zealand white rabbits by intra- and subcutaneous injection. Animals were boostered every two weeks and bled 7 days after each injection. Radioiodination Rat IAPP-15 was radioiodinated bv the lactoneroxidase method (4). The 12’1- labeled peptide was purified by reverse- phase HPLC on a TiK ODS 120A column. RIA procedure Rat IAPP[l- 371 purified from normal rat pancreas was used as a standard peptide for RIA. The incubation buffer and procedure for RIA were the same as those reported in RIA for human IAPP (5). Isolation of immunoreactive IAPP in pancreas Pancreata (7.8 g wet weight) were collected from 8 Sprague- Dawley rats after overnight fasting. The tissue was heated at 95- 100 “C for 10 min in 10 vol water. After cooling to 4 “C , CH3COOH and HCl were added to make a final concentration of 1M and 20mM, respectively, then homogenized by a Polytron for 10 min. The homogenate was centrifuged at 32,000 x g for 30 min. Two volumes of acetone were gradually added to the resulting supernatant and stirred for 12 hr at 4°C . After removing the precipitate by centrifugation at 24,000 x g for 30 min, the supernatant was evaporated and filtered through a GF/B filter (Whatman). The sample was pumped up to an octadesyl- silica column (90 ml, Chemcosorb LC-SORB SPW -C-ODS), washed with 0.5M acetic acid and 0.1% trifluoroacetic acid (TFA), then eluted with a 60% acetonitrile (CH3CN) solution containing 0.1% TFA. The eluate was evaporated and subjected to gel filtration on a Sephadex G- 50 fine column (1.8 x 134 cm, Pharmacia), using 1M acetic acid as an elution solvent. All fractions were monitored by RIA for rat IAPP. Fractions #43- 46 and #50- 55 containing immunoreactive (ir)- IAPP were pooled and lyophilized. Samples were then dissolved in O.lM sodium phosphate buffer (pH 7.4) containing 0.05% Triton X-100, and subjected to immunoaffinity chromatography on an anti-rat IAPP IgG-Affi-Gel 10 column (see below). After washing the immunoaffinity column with the above phosphate buffer, adsorbed materials were eluted with a solution of 1M acetic acid containing 10% CH3CN and 0.002% Triton X-100. Column eluate was further subjected to reverse- phase HPLC on a diphenyl column (4.6 x 250 mm,Vydac). Chromatographic conditions are described in Fig. 3 legend. All fractions were monitored by RIA. Rat IAPPs thus purified as a single peak were submitted to sequencing analyses. Immunoaffinity chromatography Immunoglobulin G (IgG) fraction was purified from 2 ml of antiserum #212- 5 by Protein A- Sepharose CL- 4B (Pharmacia). IgG was coupled with 2.5 ml of Affi- Gel 10 resin (BioRad) according to manufacturer’s instructions. More than 95% of IgG coupled with the resin. Samples were dissolved in O.lM sodium phosphate buffer @H 7.4) containing 0.05% Triton X100, loaded on the column, washed with the same phosphate buffer, then adsorbed materials were eluted with a solution of 1M acetic acid containing 10% CH3CN and 0.002% Triton X100. Capacity of the immunoaffinity resin to bind rat IAPP[l- 371 was 10 fl g/O.1 ml gel. 789
Vol. 169, No. 2, 1990
BIOCHEMICAL
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RESEARCH COMMUNICATIONS
Sequenceanalysis Sequence analysis was perfo.rmed by a gas- phase sequencer(Model 470A, Applied Biosystems).The resulting PTH- amino acids were analyzed by reverse- phaseHPLC (Model 120A, Applied Biosystems),linked with the sequencer. PTH- amino acids were measurableat concentrationsas low as0.5 pmol, and 25 pmol of a standardPTH- amino acid mixture (Pierce) was routinely usedasa calibration mixture. Determination of tissuecontent Tissueslisted in Table 1 were obtained after decapitation from 3 male Sprague-Dawley rats weighing 230- 250 g that hadbeen fed a diet of standardrat chow ad libitum. Tissueswere immediately boiled after dissection, homogenized,and centrifuged as describedabove. Each tissuecorrespondingto 100 mg wet weight, except for 3 mg of pancreas,was applied to a SepPak C- 18 cartridge and adsorbedmaterialswere eluted with 60% CH3CN solution containing 0.1% TFA. Column eluate was evaporated,subjectedto rat IAPP immunoaffinity column and eluted. The eluatewas lyophilized andreconstitutedwith RIA buffer for submissionto RIA. Characterization of rat IAPP moiety in fed and fasted rat Male Sprague- Dawley rats of 250- 300 g were divided into 2 groups: 1) fed ad lib (n=3) and 2) fasted for 4 days (n=3). Pancreaswas extracted as describedabove. Pancreatic extract correspondingto 3 mg wet tissueweight was applied to a Sep- Pak C-18 cartridge and then separatedby reverse- phaseHPLC on a TSK ODS SIL 120A column as shown in Fig. 6. Each fraction was monitoredby RIA.
RESULTS RIA for rat IAPP The subsequenceof rat IAPP from the 24th to 37th position was quite different from that of rat CGRP except for identical 6 residues.In order to obtain antibodiesspecific for rat IAPP, we synthesizedrat IAPP-15, a pentadecapeptidecorrespondingto rat IAPP[24- 371with an Nterminal arginine incorporated for solubilizing the peptide completely. Antiserum #215- 9 thus prepared was usable at a final dilution of 165,000. Rat IAPP[lpeptide for RIA since rat IAPP[l-
371 and rat IAPP-15 were equally recognized on a molar
211 pe!Xide
371 was used as a standard
Fraction
(fmolltube)
F&J:
Number
Standard curve of RIA for rat IAPP[l- 371 and cross- reactivity of antiserum. Inhibition of ~EI- rat IAPP- 15 binding to antiserum by serial dilutions of rat IAPP[l- 37]( l ), rat IAPP- 15 ( 0 ), human IAPP[ 1 - 37]( q ), rat pancreatic extract (A ), and rat CGRPs (MlF&& Sephadex G- 50 gel filtration of 7.8 g of rat pancreatic extract. Column: Sephadex G- 50, fine (1.8 x 134 cm, Pharmacia). Flow rate: 6 ml/hr. Fraction size: 5 ml/tube. Solvent: 1M acetic acid. All fractions were monitored by RIA. Black bars represent IAPP- immunoreactivity. 790
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F&J: Reverse-phaseHPLCof immunoreactive IAPP fractionisolatedby anti- rat IAPP IgG immunoaffinitychromatography. Sample:(A) eluatefromimmunoaffinitychromatography of fractions#43- 46 in Fig. 2 (B) eluatefrom immunoaffinitychromatography of fractions#50- 55 in Fig. 2 Column:219TP54 diphenyl(4.6 X 250mm,Vydac). Flow rate: 1.0ml/min. Lineargradientfrom 10%CH3CNto 60%CH3CNin 0.1%TFA for 80min.Black bars represent IAPP- immunoreactivity.Arrows indicateelutionpositionsof (1) authenticrat IAPP[l- 371and(2) rat IAPP[19- 371generated by tryptic digestionof rat IAPP[l- 371.
basisby the antiserumas shown in Fig. 1. Half- maximum inhibition by rat IAPP[l-
371was
observed at 16 fmolltube, and the peptide was detectable at a low level of 2 fmol/tube. Rat IAPP[19- 371which was isolated from rat pancreasin the presentstudy showed 100% crossreactivity with the antiserum.The antiserumexhibited very low (0.06%) cross- reactivity with human IAPP[l-
371, and no cross- reactivity with rat CGRPs. The dilution curve of rat
pancreatic extract was parallel to the standardcurve. Identification of immunoreactive IAPP in Bancreas Rat pancreatic extract was first applied to a preparative C-18 reverse- phasecolumn. Peptidesadsorbedon the column were subjectedto SephadexG- 50 gel filtration. As shown in Fig. 2, two major immunoreactive peaks(fractions #43- 46 and #50- 55) were observedon the chromatogram.Approximate molecular weights (Mr) of thesetwo componentswere estimated to be 4,000 and 2,000, respectively, basedon their elution positions.Fractions #43- 46 and#5055 were each pooled, lyophilized, and then loaded onto an anti- rat IAPP IgG immunoaffinity column. The column was thoroughly washed with O.lM sodium phosphatebuffer containing 0.05% Triton X- 100, then adsorbedpeptideswere eluted. Rat IAPPs were finally purified as a single peak by reverse- phaseHPLC on a diphenyl column as shownin Fig. 3. Yield of IAPP of Mr 4,000 and that of Mr 2,000 was 360 pmol and 720 pmol, respectively. Rat IAPP of Mr 4,000 emerged at an elution position exactly identical to that of authentic rat IAPP[lamino acid sequencinganalysishasbeen reported(3).
371 whose
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2, 1990
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5 RatIAPP[l-371
RESEARCH
10
15
20
Human
0
10 Cycle
19
Number
25
30
35
KCNTATCATQRLANFLVRSSNNLGPVLPPTNVGSNTY-NH SSNNLGPVLPPTNVGSNTY-Nk!z
Rat IAPP(W371 HumanIAPP[l-371
COMMUNICATIONS
KCNTATCATIIRLANFLVHSSNNFGAILSSTNVGSNTY-NH VHSSNNFGAILSSTNVGSNTY-NH2
IAPP[17-371
05
u:
Yield of PTH- amino acid at each cycle of Edman degradation. Sample: Peptide exhibiting IAPP- immunoreactivity in Fig. 3B. One- letter amino acid notation is used.
F&J:
Alignment of amino acid sequences of rat IAPP[lhuman IAPP[l- 371, and human IAPP[17- 371.
371, rat IAPP[19-
371,
Rat IAPP of MI 2,000 emerged15 min earlier than rat IAPP[l- 371 asshown in Fig. 3B. Stepwise Edman degradationof rat IAPP of Mr 2,000 (100 pmol) was performed by a gasphasesequencerand the PTH- amino acids liberated were identified up to the C- terminal residue of 19- amino acids as shown in Fig. 4. The amino acid sequenceof the IAPP thus determined is demonstratedin Fig. 5. The peptide was found to correspondto the subsequence [19- 371 of rat IAPP. C- terminal amidation of the IAPP was confirmed basedon its elution position identical to that of rat IAPP[19- 371generatedby tryptic digestion of rat IAPP[l-
371
asshown in Fig. 3B. Tissuecontent of IAPP Regional distribution of IAPP in fed rats is presentedin Table 1. Tissue sampleswere extracted through a Sep-Pak C-18 cartridge and then immunoaffinity column in order to avoid
Table 1. Tissue content of rat IAPP tissue pancreas fed fasted esophagus stomach esophageal region pyloric antrum duodenum jejunum ileum colon
pmol/g wet weight 328.5 k 178.9 + CO.1 CO.1 2.5 + 0.2 + 0.4 f 0.4 * 1.2 f
25.0 38.9
0 0.2 0 0.1 0.2
tissue heart lung liver spleen kidney testis cerebrum cerebellum pituitary
pmol/g wet weight CO.1
Vol. 169, No. 2, 1990 June 15, 1990
REGIONAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 788-795
DISTRIBUTION AND MOLECULAR FORMS OF RAT ISLET AMYLOID POLYPEPTIDE
Junko ASAIl, MasamitsuNAKAZATO’O, Mikiya MIYAZATO’, Kenji KANGAWA2, Hisayuki MATSU03 and Shigeru MATSUKURA’ Departmentsof ‘Internal Medicine and ‘Biochemistry, Miyazaki Medical College, Kiyotake, Miyazaki 889- 16, Japan 3National CardiovascularCenter ResearchInstitute, Suita, Osaka565, Japan Received
May 7, 1990
SUMMARY: Using a highly sensitive and specific radioimmunoassay(RIA) for rat islet amyloid polypeptide (IAPP), we clarified regional distribution and molecular forms of rat IAPP. IAPP[l- 371 and IAPP[19- 371were identified in normal rat pancreasby sequenceanalyses. IAPP[19- 371, accounting for 57% of IAPP- immunoreactivity in rat pancreas,is a major molecular form of rat IAPP moiety. In human,however, IAPP[l- 371 is the major component, with IAPP[17- 371composingas little as 2- 6% of IAPP- immunoreactivity in pancreas.This indicatesthat processingof IAPP in pancreasdiffers in species.A large amount of IAPP (328.5 f 25.0 pmollg wet weight) was found in rat pancreasand the peptide was also detected in pyloric antrum of the stomach,duodenum,jejunum, ileum, and colon at O.l- 0.8% of the level of pancreas. It was not detected in central nervoussystem. The content of rat IAPP in pancreas fell to 54% of control after 4 day fasting. The distribution of IAPP suggestsits possible endocrineor paracrinefunction in pancreasand gastrointestinaltract. 01990 Academic Press, I~C.
Islet amyloid polypeptide (IAPP) (l), also designatedamylin (2) is a 37- amino acid polypeptide which constitutesislet amyloid in pancreasof patients with non-insulin- dependent diabetesmellitus. IAPP is a member of the calcitonin gene- related peptide (CGRP) family. Amino acid sequencesof the two peptides are 46% identical. We have recently isolated rat IAPP from normal rat pancreasby utilizing the cross- reactivity of an antiserumraisedagainst human IAPP with rat IAPP (3). Both rat and human IAPPs are 37 amino acids long and their sequencesare 84% identical. In the present study, we establisheda sensitive and specific radioimmunoassay(RIA) system for rat IAPP basedon its sequence.Using RIA, we studied regional distribution of rat IAPP to delineate its physiological function. Furthermore, we identified IAPP[l - 371 and §To whom correspondenceshouldbe addressed. ABBREVIATIONS: CGRP, calcitonin gene- related peptide. HPLC, high performance liquid chromatography. w, islet amyloid polypeptide. k, immunoreactive. m, radioimmunoassay. 0006-291X/90 Copyright All rights
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
788
Vol.
169,
IAPP[19-
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
371 in rat pancreas by sequence analyses. Changes in the content and molecular forms
of rat IAPP in pancreas in response to fasting were also demonstrated.
MATERIALS
AND METHODS
Peptide synthesis A pentadecapeptide, termed rat IAPP-15 which corresponds to subsequence [24- 371 of rat IAPP with an additional arginine at the N- terminus, was synthesized by solid phase techniques, using peptide synthesizer Model 430A (Applied Biosystems). The peptide was purified by reverse- phase high performance liquid chromatography (HPLC) on a p - Bondasphere 5 fi C18 300A column. Correct synthesis was confirmed by amino acid analysis and sequencing. Preparation of antiserum Rat IAPP -15 (12 mg) was conjugated with bovine thyroglobulin (16 mg) by the glutaraldehyde method. The reaction mixture was dialyzed four times against one liter of 0.9% NaCl. The antigenic conjugate solution (1.5- 3 ml) was emulsified with an equal volume of Freund’s complete adjuvant, and used for immunizing New Zealand white rabbits by intra- and subcutaneous injection. Animals were boostered every two weeks and bled 7 days after each injection. Radioiodination Rat IAPP-15 was radioiodinated bv the lactoneroxidase method (4). The 12’1- labeled peptide was purified by reverse- phase HPLC on a TiK ODS 120A column. RIA procedure Rat IAPP[l- 371 purified from normal rat pancreas was used as a standard peptide for RIA. The incubation buffer and procedure for RIA were the same as those reported in RIA for human IAPP (5). Isolation of immunoreactive IAPP in pancreas Pancreata (7.8 g wet weight) were collected from 8 Sprague- Dawley rats after overnight fasting. The tissue was heated at 95- 100 “C for 10 min in 10 vol water. After cooling to 4 “C , CH3COOH and HCl were added to make a final concentration of 1M and 20mM, respectively, then homogenized by a Polytron for 10 min. The homogenate was centrifuged at 32,000 x g for 30 min. Two volumes of acetone were gradually added to the resulting supernatant and stirred for 12 hr at 4°C . After removing the precipitate by centrifugation at 24,000 x g for 30 min, the supernatant was evaporated and filtered through a GF/B filter (Whatman). The sample was pumped up to an octadesyl- silica column (90 ml, Chemcosorb LC-SORB SPW -C-ODS), washed with 0.5M acetic acid and 0.1% trifluoroacetic acid (TFA), then eluted with a 60% acetonitrile (CH3CN) solution containing 0.1% TFA. The eluate was evaporated and subjected to gel filtration on a Sephadex G- 50 fine column (1.8 x 134 cm, Pharmacia), using 1M acetic acid as an elution solvent. All fractions were monitored by RIA for rat IAPP. Fractions #43- 46 and #50- 55 containing immunoreactive (ir)- IAPP were pooled and lyophilized. Samples were then dissolved in O.lM sodium phosphate buffer (pH 7.4) containing 0.05% Triton X-100, and subjected to immunoaffinity chromatography on an anti-rat IAPP IgG-Affi-Gel 10 column (see below). After washing the immunoaffinity column with the above phosphate buffer, adsorbed materials were eluted with a solution of 1M acetic acid containing 10% CH3CN and 0.002% Triton X-100. Column eluate was further subjected to reverse- phase HPLC on a diphenyl column (4.6 x 250 mm,Vydac). Chromatographic conditions are described in Fig. 3 legend. All fractions were monitored by RIA. Rat IAPPs thus purified as a single peak were submitted to sequencing analyses. Immunoaffinity chromatography Immunoglobulin G (IgG) fraction was purified from 2 ml of antiserum #212- 5 by Protein A- Sepharose CL- 4B (Pharmacia). IgG was coupled with 2.5 ml of Affi- Gel 10 resin (BioRad) according to manufacturer’s instructions. More than 95% of IgG coupled with the resin. Samples were dissolved in O.lM sodium phosphate buffer @H 7.4) containing 0.05% Triton X100, loaded on the column, washed with the same phosphate buffer, then adsorbed materials were eluted with a solution of 1M acetic acid containing 10% CH3CN and 0.002% Triton X100. Capacity of the immunoaffinity resin to bind rat IAPP[l- 371 was 10 fl g/O.1 ml gel. 789
Vol. 169, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Sequenceanalysis Sequence analysis was perfo.rmed by a gas- phase sequencer(Model 470A, Applied Biosystems).The resulting PTH- amino acids were analyzed by reverse- phaseHPLC (Model 120A, Applied Biosystems),linked with the sequencer. PTH- amino acids were measurableat concentrationsas low as0.5 pmol, and 25 pmol of a standardPTH- amino acid mixture (Pierce) was routinely usedasa calibration mixture. Determination of tissuecontent Tissueslisted in Table 1 were obtained after decapitation from 3 male Sprague-Dawley rats weighing 230- 250 g that hadbeen fed a diet of standardrat chow ad libitum. Tissueswere immediately boiled after dissection, homogenized,and centrifuged as describedabove. Each tissuecorrespondingto 100 mg wet weight, except for 3 mg of pancreas,was applied to a SepPak C- 18 cartridge and adsorbedmaterialswere eluted with 60% CH3CN solution containing 0.1% TFA. Column eluate was evaporated,subjectedto rat IAPP immunoaffinity column and eluted. The eluatewas lyophilized andreconstitutedwith RIA buffer for submissionto RIA. Characterization of rat IAPP moiety in fed and fasted rat Male Sprague- Dawley rats of 250- 300 g were divided into 2 groups: 1) fed ad lib (n=3) and 2) fasted for 4 days (n=3). Pancreaswas extracted as describedabove. Pancreatic extract correspondingto 3 mg wet tissueweight was applied to a Sep- Pak C-18 cartridge and then separatedby reverse- phaseHPLC on a TSK ODS SIL 120A column as shown in Fig. 6. Each fraction was monitoredby RIA.
RESULTS RIA for rat IAPP The subsequenceof rat IAPP from the 24th to 37th position was quite different from that of rat CGRP except for identical 6 residues.In order to obtain antibodiesspecific for rat IAPP, we synthesizedrat IAPP-15, a pentadecapeptidecorrespondingto rat IAPP[24- 371with an Nterminal arginine incorporated for solubilizing the peptide completely. Antiserum #215- 9 thus prepared was usable at a final dilution of 165,000. Rat IAPP[lpeptide for RIA since rat IAPP[l-
371 and rat IAPP-15 were equally recognized on a molar
211 pe!Xide
371 was used as a standard
Fraction
(fmolltube)
F&J:
Number
Standard curve of RIA for rat IAPP[l- 371 and cross- reactivity of antiserum. Inhibition of ~EI- rat IAPP- 15 binding to antiserum by serial dilutions of rat IAPP[l- 37]( l ), rat IAPP- 15 ( 0 ), human IAPP[ 1 - 37]( q ), rat pancreatic extract (A ), and rat CGRPs (MlF&& Sephadex G- 50 gel filtration of 7.8 g of rat pancreatic extract. Column: Sephadex G- 50, fine (1.8 x 134 cm, Pharmacia). Flow rate: 6 ml/hr. Fraction size: 5 ml/tube. Solvent: 1M acetic acid. All fractions were monitored by RIA. Black bars represent IAPP- immunoreactivity. 790
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(mini
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F&J: Reverse-phaseHPLCof immunoreactive IAPP fractionisolatedby anti- rat IAPP IgG immunoaffinitychromatography. Sample:(A) eluatefromimmunoaffinitychromatography of fractions#43- 46 in Fig. 2 (B) eluatefrom immunoaffinitychromatography of fractions#50- 55 in Fig. 2 Column:219TP54 diphenyl(4.6 X 250mm,Vydac). Flow rate: 1.0ml/min. Lineargradientfrom 10%CH3CNto 60%CH3CNin 0.1%TFA for 80min.Black bars represent IAPP- immunoreactivity.Arrows indicateelutionpositionsof (1) authenticrat IAPP[l- 371and(2) rat IAPP[19- 371generated by tryptic digestionof rat IAPP[l- 371.
basisby the antiserumas shown in Fig. 1. Half- maximum inhibition by rat IAPP[l-
371was
observed at 16 fmolltube, and the peptide was detectable at a low level of 2 fmol/tube. Rat IAPP[19- 371which was isolated from rat pancreasin the presentstudy showed 100% crossreactivity with the antiserum.The antiserumexhibited very low (0.06%) cross- reactivity with human IAPP[l-
371, and no cross- reactivity with rat CGRPs. The dilution curve of rat
pancreatic extract was parallel to the standardcurve. Identification of immunoreactive IAPP in Bancreas Rat pancreatic extract was first applied to a preparative C-18 reverse- phasecolumn. Peptidesadsorbedon the column were subjectedto SephadexG- 50 gel filtration. As shown in Fig. 2, two major immunoreactive peaks(fractions #43- 46 and #50- 55) were observedon the chromatogram.Approximate molecular weights (Mr) of thesetwo componentswere estimated to be 4,000 and 2,000, respectively, basedon their elution positions.Fractions #43- 46 and#5055 were each pooled, lyophilized, and then loaded onto an anti- rat IAPP IgG immunoaffinity column. The column was thoroughly washed with O.lM sodium phosphatebuffer containing 0.05% Triton X- 100, then adsorbedpeptideswere eluted. Rat IAPPs were finally purified as a single peak by reverse- phaseHPLC on a diphenyl column as shownin Fig. 3. Yield of IAPP of Mr 4,000 and that of Mr 2,000 was 360 pmol and 720 pmol, respectively. Rat IAPP of Mr 4,000 emerged at an elution position exactly identical to that of authentic rat IAPP[lamino acid sequencinganalysishasbeen reported(3).
371 whose
Vol.
169,
No.
2, 1990
BIOCHEMICAL
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BIOPHYSICAL
5 RatIAPP[l-371
RESEARCH
10
15
20
Human
0
10 Cycle
19
Number
25
30
35
KCNTATCATQRLANFLVRSSNNLGPVLPPTNVGSNTY-NH SSNNLGPVLPPTNVGSNTY-Nk!z
Rat IAPP(W371 HumanIAPP[l-371
COMMUNICATIONS
KCNTATCATIIRLANFLVHSSNNFGAILSSTNVGSNTY-NH VHSSNNFGAILSSTNVGSNTY-NH2
IAPP[17-371
05
u:
Yield of PTH- amino acid at each cycle of Edman degradation. Sample: Peptide exhibiting IAPP- immunoreactivity in Fig. 3B. One- letter amino acid notation is used.
F&J:
Alignment of amino acid sequences of rat IAPP[lhuman IAPP[l- 371, and human IAPP[17- 371.
371, rat IAPP[19-
371,
Rat IAPP of MI 2,000 emerged15 min earlier than rat IAPP[l- 371 asshown in Fig. 3B. Stepwise Edman degradationof rat IAPP of Mr 2,000 (100 pmol) was performed by a gasphasesequencerand the PTH- amino acids liberated were identified up to the C- terminal residue of 19- amino acids as shown in Fig. 4. The amino acid sequenceof the IAPP thus determined is demonstratedin Fig. 5. The peptide was found to correspondto the subsequence [19- 371 of rat IAPP. C- terminal amidation of the IAPP was confirmed basedon its elution position identical to that of rat IAPP[19- 371generatedby tryptic digestion of rat IAPP[l-
371
asshown in Fig. 3B. Tissuecontent of IAPP Regional distribution of IAPP in fed rats is presentedin Table 1. Tissue sampleswere extracted through a Sep-Pak C-18 cartridge and then immunoaffinity column in order to avoid
Table 1. Tissue content of rat IAPP tissue pancreas fed fasted esophagus stomach esophageal region pyloric antrum duodenum jejunum ileum colon
pmol/g wet weight 328.5 k 178.9 + CO.1 CO.1 2.5 + 0.2 + 0.4 f 0.4 * 1.2 f
25.0 38.9
0 0.2 0 0.1 0.2
tissue heart lung liver spleen kidney testis cerebrum cerebellum pituitary
pmol/g wet weight CO.1
