Peptides. Vol. 11, pp. 683--685. ©Pergamon Press plc, 1990. Printed in the U.S.A.
0196-9781/90 $3.00 + .00
Purification of Peptide Hormones From Chinchilla Pancreas by Chemical Assay J. E N G , * t 1 W. A. K L E I N M A N * A N D L . - S . C H U *
*Solomon A. Berson Research Laboratory, Veterans Affairs Medical Center, Bronx, NY 10468 fMt. Sinai School of Medicine, CUNY, New York, N Y 10029 R e c e i v e d 13 February 1990
ENG, J., W. A. KLEINMAN AND L.-S. CHU. Purification of peptide hormonesfrom chinchilla pancreas by chemical assay. PEPTIDES 11(4) 683~685, 1990.--Glucagon was purified from chinchilla pancreas and its biological activity determined. It was isolated using a chemical assay to identify peptides with a histidyl residue at the N-terminus. Chinchilla glucagon has the amino acid sequence HSQGTFTSDYSKHLDSRYAQEFVQWLMNT. It differs from the usual mammalian glucagon by amino acid substitutions at positions 13, 18 and 21 from the N-terminus. Despite these sequence changes, its biological activity is conserved. Chinchilla glucagon has approximately the same potency as pig glucagon in stimulating liver membrane adenyl cyclase activity. Pancreatic polypeptide was also purified from chinchilla pancreas based on its Ala l signal and has the sequence APLEPVYPGDNATPEQMAQYAAEMRRYINMLTRPRY#. Purification
Chinchilla
Chemical assay
Amino acid sequence
Glucagon
Pancreatic polypeptide
Pancreatic tissue with a combined weight of 8 grams was dissected from six chinchillas. The tissue was extracted in 10 volumes acid-alcohol (75% ethanol-0.2 M HCI). The extract was centrifuged and the supernate was mixed with 8 volumes acetone. The resulting precipitate was collected by centrifugation and dissolved in 1 M acetic acid. The pancreatic peptides contained in the acetic acid solution were concentrated by passage through a C18 Sep-Pak (Waters Associates, Milford, MA) and eluted from the cartridge with 2 ml of 0.1% trifluoroacetic acid (TFA)/60% acetonitrile (ACN) prior to HPLC separation. Peptide purifications were monitored with single cycle amino acid sequencing using an automated gas-phase sequencer with an on-line phenylthiohydantoin amino acid analyzer (Applied Biosystems, Foster City, CA). Portions of purified peptides (100--400 pmol) were separately subjected to chemical or enzymatic cleavage. Chinchilla PP was chemically cleaved with 0.1 ml 50% TFA containing 18 mg cyanogen bromide. Enzyme cleavages of glucagon were carded out in 0.1 ml 100 mM ammonium bicarbonate containing either 0.003 unit Endoproteinase Lys-C, 0.1 unit Endoproteinase Arg-C or 0.25 ixg Endoproteinase Glu-C. Insulin was cleaved into its A and B chains by a reduction-alkylation procedure as previously described (1). The peptide chains and fragments from these reactions were separately purified by reverse-phase HPLC. Intact peptides or peptide fragments were sequenced with the gas-phase sequencer.
THE New World hystricomorph rodents, guinea pig and chinchilla, share common sequence changes in several peptide hormones which differ uniquely from those of other mammals. These peptides include gastrin (3,9) and cholecystokinin (7,13). Earlier studies had shown that guinea pig glucagon has an altered C-terminal amino acid sequence and a 10-fold lower potency in a liver receptor binding assay (5,8). Our observation that extracts of chinchilla pancreas had little or no immunoreactivity when tested with several glucagon antisera available in this laboratory suggested that chinchilla glucagon also differs from the usual mammalian glucagon. We postulated that chinchilla glucagon, despite its poor reactivity with glucagon antisera, was likely to retain the N-terminal histidyl (His l) residue that is characteristic of peptides in the glucagon-secretin-VIP family and that N-terminal sequence analysis could be used to detect this peptide in pancreatic extracts. In this study, we report the use of N-terminal sequence analysis as a chemical assay to monitor peptides for the presence of His I in the purification of chinchilla glucagon. N-terminal analysis also detected the N-terminal amino acids of chinchilla PP and insulin which permitted their simultaneous purification. METHOD Frozen chinchilla carcasses with pelts removed were obtained from Chinchilla of Catskill (Catskill, NY). Endoproteinases were purchased from Boehringer-Mannheim (Indianapolis, IN).
1Requests for reprints should be addressed to John Eng, Solomon A. Berson Research Laboratory, Veterans Affairs Medical Center, 130 W. Kingsbridge Road, Bronx, NY 10468.
683
684
ENG, KLEINMAN AND CHU
Purified chinchilla glucagon was assayed for its ability to activate adenyl cyclase in a liver membrane preparation (2). Liver membranes were prepared by homogenizing pieces of dog liver in a loose-fitting glass homogenizer with Teflon pestle in 5 volumes 1 mM sodium bicarbonate. Large tissue debris was removed from the suspension by passage across a mesh filter. The membrane suspension was centrifuged at 10,000 x g at 4°C for 30 minutes. The supernate was removed and the pellet resuspended in 10 volumes 1 mM sodium bicarbonate according to the original tissue weight. The membrane suspension was stored frozen in aliquots at - 7 0 ° C until use. Chinchilla or pig glucagon at increasing concentrations were incubated in a solution containing 2 mM ATP, 0.1 mM GTP, 5 mM MgC12, 1 mM IBMX, 1 mg/ml bacitracin, 1 mg/ml bovine albumin and 0.2 mg wet weight dog liver membrane in 50 mM Tris-HCl, pH 7.4. Adenyl cyclase activity was determined by the amount of cyclic A M P generated from ATP over a 60-minute period. Cyclic AMP was measured by radioimmunoassay (10).
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0196-9781/90 $3.00 + .00
Purification of Peptide Hormones From Chinchilla Pancreas by Chemical Assay J. E N G , * t 1 W. A. K L E I N M A N * A N D L . - S . C H U *
*Solomon A. Berson Research Laboratory, Veterans Affairs Medical Center, Bronx, NY 10468 fMt. Sinai School of Medicine, CUNY, New York, N Y 10029 R e c e i v e d 13 February 1990
ENG, J., W. A. KLEINMAN AND L.-S. CHU. Purification of peptide hormonesfrom chinchilla pancreas by chemical assay. PEPTIDES 11(4) 683~685, 1990.--Glucagon was purified from chinchilla pancreas and its biological activity determined. It was isolated using a chemical assay to identify peptides with a histidyl residue at the N-terminus. Chinchilla glucagon has the amino acid sequence HSQGTFTSDYSKHLDSRYAQEFVQWLMNT. It differs from the usual mammalian glucagon by amino acid substitutions at positions 13, 18 and 21 from the N-terminus. Despite these sequence changes, its biological activity is conserved. Chinchilla glucagon has approximately the same potency as pig glucagon in stimulating liver membrane adenyl cyclase activity. Pancreatic polypeptide was also purified from chinchilla pancreas based on its Ala l signal and has the sequence APLEPVYPGDNATPEQMAQYAAEMRRYINMLTRPRY#. Purification
Chinchilla
Chemical assay
Amino acid sequence
Glucagon
Pancreatic polypeptide
Pancreatic tissue with a combined weight of 8 grams was dissected from six chinchillas. The tissue was extracted in 10 volumes acid-alcohol (75% ethanol-0.2 M HCI). The extract was centrifuged and the supernate was mixed with 8 volumes acetone. The resulting precipitate was collected by centrifugation and dissolved in 1 M acetic acid. The pancreatic peptides contained in the acetic acid solution were concentrated by passage through a C18 Sep-Pak (Waters Associates, Milford, MA) and eluted from the cartridge with 2 ml of 0.1% trifluoroacetic acid (TFA)/60% acetonitrile (ACN) prior to HPLC separation. Peptide purifications were monitored with single cycle amino acid sequencing using an automated gas-phase sequencer with an on-line phenylthiohydantoin amino acid analyzer (Applied Biosystems, Foster City, CA). Portions of purified peptides (100--400 pmol) were separately subjected to chemical or enzymatic cleavage. Chinchilla PP was chemically cleaved with 0.1 ml 50% TFA containing 18 mg cyanogen bromide. Enzyme cleavages of glucagon were carded out in 0.1 ml 100 mM ammonium bicarbonate containing either 0.003 unit Endoproteinase Lys-C, 0.1 unit Endoproteinase Arg-C or 0.25 ixg Endoproteinase Glu-C. Insulin was cleaved into its A and B chains by a reduction-alkylation procedure as previously described (1). The peptide chains and fragments from these reactions were separately purified by reverse-phase HPLC. Intact peptides or peptide fragments were sequenced with the gas-phase sequencer.
THE New World hystricomorph rodents, guinea pig and chinchilla, share common sequence changes in several peptide hormones which differ uniquely from those of other mammals. These peptides include gastrin (3,9) and cholecystokinin (7,13). Earlier studies had shown that guinea pig glucagon has an altered C-terminal amino acid sequence and a 10-fold lower potency in a liver receptor binding assay (5,8). Our observation that extracts of chinchilla pancreas had little or no immunoreactivity when tested with several glucagon antisera available in this laboratory suggested that chinchilla glucagon also differs from the usual mammalian glucagon. We postulated that chinchilla glucagon, despite its poor reactivity with glucagon antisera, was likely to retain the N-terminal histidyl (His l) residue that is characteristic of peptides in the glucagon-secretin-VIP family and that N-terminal sequence analysis could be used to detect this peptide in pancreatic extracts. In this study, we report the use of N-terminal sequence analysis as a chemical assay to monitor peptides for the presence of His I in the purification of chinchilla glucagon. N-terminal analysis also detected the N-terminal amino acids of chinchilla PP and insulin which permitted their simultaneous purification. METHOD Frozen chinchilla carcasses with pelts removed were obtained from Chinchilla of Catskill (Catskill, NY). Endoproteinases were purchased from Boehringer-Mannheim (Indianapolis, IN).
1Requests for reprints should be addressed to John Eng, Solomon A. Berson Research Laboratory, Veterans Affairs Medical Center, 130 W. Kingsbridge Road, Bronx, NY 10468.
683
684
ENG, KLEINMAN AND CHU
Purified chinchilla glucagon was assayed for its ability to activate adenyl cyclase in a liver membrane preparation (2). Liver membranes were prepared by homogenizing pieces of dog liver in a loose-fitting glass homogenizer with Teflon pestle in 5 volumes 1 mM sodium bicarbonate. Large tissue debris was removed from the suspension by passage across a mesh filter. The membrane suspension was centrifuged at 10,000 x g at 4°C for 30 minutes. The supernate was removed and the pellet resuspended in 10 volumes 1 mM sodium bicarbonate according to the original tissue weight. The membrane suspension was stored frozen in aliquots at - 7 0 ° C until use. Chinchilla or pig glucagon at increasing concentrations were incubated in a solution containing 2 mM ATP, 0.1 mM GTP, 5 mM MgC12, 1 mM IBMX, 1 mg/ml bacitracin, 1 mg/ml bovine albumin and 0.2 mg wet weight dog liver membrane in 50 mM Tris-HCl, pH 7.4. Adenyl cyclase activity was determined by the amount of cyclic A M P generated from ATP over a 60-minute period. Cyclic AMP was measured by radioimmunoassay (10).
Glyl/~' ,)20~
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__08
IfTf, 20
40
60
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