Regulatory Peptides, 32 (1991) 39-45 Elsevier
39
REGPEP 00982
Rhesus monkey gastroenteropancreatic hormones" relationship to human sequences Jinghua Yu l, Yan Xin
1,
John Eng 1,2 and Rosalyn S. Yalow 1'2
ISolomon A. Berson Research Laboratory, Department of Veterans Affairs Medical Center, Bronx, N Y and 2The Mount Sinai School of Medicine, CUNY, New York, N Y (U.S.A.) (Received 7 August 1990; revised version received 24 September 1990; accepted 25 September 1990)
Key words: Amino acid sequence; Insulin; Glucagon; Pancreatic polypeptide; Vasoactive intestinal polypeptide; Gastrin
Summary The amino acid sequences of the gastroenteropancreatic peptides of Old World mammals are generally well-conserved. However, only the glucagons and vasoactive intestinal polypeptides (VIP) have been shown to be identical among the species studied to date. Rhesus monkey (Macaca mulana) insulin has been shown to be identical with human insulin. The question addressed in this study is whether other gastroenteropancreatic peptides are identical to the human peptides. Purification and sequencing of glucagon, pancreatic polypeptide, VIP and insulin confirmed their identity with the corresponding human peptides. However, the 17 amino acid monkey gastrin is identical to dog gastrin and differs from human gastrin by substitution of methionine for leucine at position 5 from the N-terminus and alanine for glutamic acid in position 10. If additional rhesus monkey tissues become available, it would be of interest to determine whether other gastrointestinal peptides also differ from the corresponding human peptides.
Introduction The sequences of gastroenteropancreatic peptides of Old World mammals are generally well-conserved. Thus, the insulins of several species differ at only one of four
Correspondence: R.S. Yalow, Solomon A. Berson Research Laboratory, VA Medical Center, 130 West Kingsbridge Road, Bronx, NY 10468, U.S.A. 0167-0115/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
4()
positions among the 51 amino acids of the A and B chains [1 ]. The glucagons [2-6] and vasoactive intestinal polypeptides (VIP) [7-10] have been identical among the species of Old World mammals studied to date. The pancreatic polypeptides (PP) differ in 4 of 36 sites [ 11 ] and the gastrins differ in 2 of 17 sites [ 12,13 ]. The insulin of the Old World monkey (Macaca mulatta) has been shown to be identical to human insulin [ 14]. While it was expected that rhesus monkey glucagon and VIP would be identical to the human peptides, a question of interest is whether other hormonal peptides would also be identical to the corresponding human peptides. In this report, we describe the purification and sequencing of five peptides of the rhesus monkey gastroenteropancreatic axis and demonstrate that only one of the five, gastrin, differs from the human peptide.
Materials and Methods
Rhesus monkey (Macaca mulatta) tissues were obtained from the Yerkes Regional Primate Research Center at Emory University, Atlanta, GA. The tissues were collected shortly after death and were maintained frozen until extracted for the various peptides. Steps in the purification of the five peptides were monitored using in-house radioimmunoassay (RIA) procedures in general use in our laboratory, using appropriate standards for human peptides. The gastrin RIA employs an antiserum that recognizes the gastrin COOH-terminus, but does not detect the deamidated gastrin at molar concentrations 1000-fold higher than the corresponding amidated peptide [13]. One pancreas weighing 6.4 g was extracted in 5 vol. of acid-alcohol (0.2 M HC1/75 ~o ethanol) with a Teflon tissue grinder. The acid-alcohol extract was precipitated with 8 vol. of acetone at - 30 °C overnight. The precipitate was collected by centrifugation and the supernatant was discarded. The precipitate was dissolved in 8.5 ml of 1 M acetic acid and contained 309 nmol insulin, 148 nmol glucagon and 40 nmol PP. A C18 Sep-Pak cartridge (Water Associates, Milford, MA) was prepared by washing with 10 ml ethanol and 10 mt distilled water. The redissolved precipitate was then pumped through at 1 ml/min. The cartridge was washed with 6 ml 0.1 ~o trifluoroacetic acid (TFA) and eluted with 2 ml 0.1 ~o TFA/60~o acetonitrile (ACN). The eluate was further purified by HPLC. The eluate was applied to a MB C~8 radial-pak column (Waters Associates) and eluted with a linear gradient from 20-60~o ACN in 0.13~o heptafluorobutyric acid (HFBA). Following this step, the major peaks of insulin and PP were pure, but the glucagon was contaminated with insulin. The insulin was reduced and alkylated, and the A and B chains were separated by a single HPLC step using a Nova C 18 column which was eluted with a linear gradient from 0-60 ~o ACN in 0.1 ~o TFA. The MB C~8 peak containing glucagon and small amounts of insulin was reduced and alkylated. The glucagon was then separated from the A and B insulin chains by an HPLC step using a Nova C~8 column. For the purificiation of VIP the colons from four monkeys were thawed prior to removal of the mucosa by scraping. The remaining muscle (183 g total) was extracted with 5 vol. 0.1 M HC1/90~o ethanol. The acid-alcohol extract was precipitated with 3 vol. of acetone and stored overnight at - 30 °C. The precipitate that formed was
41 removed by centrifugation. Following centrifugation the supernatant was discarded. The precipitate was redissolved in 20 ml acetic acid and contained 10.5 nmol VIP. The acetic acid solution was concentrated on a C~8 Sep-Pak cartridge, which was eluted with 2 ml 0.1 ~o TFA/60 ~o ACN. The eluate containing 7.6 nmol VIP was further processed by dilution with 8 ml 0.05 M Na acetate (pH 5) and passed through a CM Sep-Pak cartridge. The adsorbed VIP was eluted with 5 ml 0.05 M Na acetate/0.5 M NaC1 (7.1 nmol). Final purification was effected by four HPLC steps. For the purificiation of gastrin the stomachs of eight monkeys were defrosted in methanol and the antral mucosa separated. The mucosa (43 g) was boiled in H20 for 5 min, cooled and then extracted in 10 vol. of 0.1 M N H n C O 3 (22 nmol). The suspension was centrifuged at 3000g for 30 min at 4°C. The supernatant was further clarified by the addition of 2 vol. of acetone and stored overnight at - 30 ° C. The precipitate that formed was removed by centrifugation. The extract was pumped through two QMA cartridges (Waters Associates) connected in series. Each cartridge was washed with 10 ml 0.05 M Tris (pH 7) (Tris) and eluted with 4 ml Tris/1 M NaC1. The combined eluates containing 14 nmol gastrin were applied to a Sephadex G50SF (450 ml) column which was eluted at a flow rate of 60 ml/h with Tris. The elution fractions containing the major peaks of gastrin were pooled, applied to a MB C18 radial-pak column and eluted with a linear gradient from 0-30~o ACN in Tris. The NH2-terminus of the major peak was enzymatically deblocked with pyroglutamyl aminopeptidase (PAP, Sigma Chemical Co.) at a ratio of 2 units PAP/nmol 'little' gastrin in a volume of 200/~1. Deblocked peptides were repurified on a Nova C~8 column prior to sequence analysis. The COOH-terminal sequence of monkey gastrin was obtained by digesting intact gastrin (0.4 nmol) with 1 #g endoproteinase Glu-C (Boehringer-Mannheim Biochemicals, Indianapolis, IN) in 0.1 M N H n C O 3 overnight at room temperature. The mixture was sequenced directly. The purified peptides (50-200 pmol) were sequenced on an automated gas-phase sequencer with an on-line PTH amino acid analyzer (Applied Biosystems, Foster City, CA).
Results
The HPLC steps in the purification of the pancreatic peptides are shown in Fig. 1. Following a single step (left) the insulin peak and the PP peak were sufficiently pure for sequencing, but the glucagon peak contained a minor insulin contamination. The insulin from the peak fraction was reduced and alkylated, and the pure A and B chains were separated by H P L C in preparation for sequence analysis (Fig. 1, top right). The A and B chains were completely sequenced and shown to be identical to the A and B chains of human insulin. Following reduction and alkylation of the insulin-contaminated glucagon peak the glucagon was separated from the contaminating A and B chains by HPLC. (Fig. 1, lower right). Direct sequencing provided the sequence of the 24 N-terminal amino acids. Following treatment with endoproteinase Arg-C, the overlapping C-terminus fragment was sequenced. The monkey glucagon was identical to the usual mammalian glucagon. The PP was completely sequenced and shown to be identical to the corresponding human peptide.
42 MB CI8
....
05
,Nqm°l~;t R~/o ql 2t7 • 72 72 3; B 26,6 76 ,
'
Nova CI8
In~m 302 Gklcogon IOO
A
.I1/./"/1
Chmn
60
.11 ././
39
REGPEP 00982
Rhesus monkey gastroenteropancreatic hormones" relationship to human sequences Jinghua Yu l, Yan Xin
1,
John Eng 1,2 and Rosalyn S. Yalow 1'2
ISolomon A. Berson Research Laboratory, Department of Veterans Affairs Medical Center, Bronx, N Y and 2The Mount Sinai School of Medicine, CUNY, New York, N Y (U.S.A.) (Received 7 August 1990; revised version received 24 September 1990; accepted 25 September 1990)
Key words: Amino acid sequence; Insulin; Glucagon; Pancreatic polypeptide; Vasoactive intestinal polypeptide; Gastrin
Summary The amino acid sequences of the gastroenteropancreatic peptides of Old World mammals are generally well-conserved. However, only the glucagons and vasoactive intestinal polypeptides (VIP) have been shown to be identical among the species studied to date. Rhesus monkey (Macaca mulana) insulin has been shown to be identical with human insulin. The question addressed in this study is whether other gastroenteropancreatic peptides are identical to the human peptides. Purification and sequencing of glucagon, pancreatic polypeptide, VIP and insulin confirmed their identity with the corresponding human peptides. However, the 17 amino acid monkey gastrin is identical to dog gastrin and differs from human gastrin by substitution of methionine for leucine at position 5 from the N-terminus and alanine for glutamic acid in position 10. If additional rhesus monkey tissues become available, it would be of interest to determine whether other gastrointestinal peptides also differ from the corresponding human peptides.
Introduction The sequences of gastroenteropancreatic peptides of Old World mammals are generally well-conserved. Thus, the insulins of several species differ at only one of four
Correspondence: R.S. Yalow, Solomon A. Berson Research Laboratory, VA Medical Center, 130 West Kingsbridge Road, Bronx, NY 10468, U.S.A. 0167-0115/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
4()
positions among the 51 amino acids of the A and B chains [1 ]. The glucagons [2-6] and vasoactive intestinal polypeptides (VIP) [7-10] have been identical among the species of Old World mammals studied to date. The pancreatic polypeptides (PP) differ in 4 of 36 sites [ 11 ] and the gastrins differ in 2 of 17 sites [ 12,13 ]. The insulin of the Old World monkey (Macaca mulatta) has been shown to be identical to human insulin [ 14]. While it was expected that rhesus monkey glucagon and VIP would be identical to the human peptides, a question of interest is whether other hormonal peptides would also be identical to the corresponding human peptides. In this report, we describe the purification and sequencing of five peptides of the rhesus monkey gastroenteropancreatic axis and demonstrate that only one of the five, gastrin, differs from the human peptide.
Materials and Methods
Rhesus monkey (Macaca mulatta) tissues were obtained from the Yerkes Regional Primate Research Center at Emory University, Atlanta, GA. The tissues were collected shortly after death and were maintained frozen until extracted for the various peptides. Steps in the purification of the five peptides were monitored using in-house radioimmunoassay (RIA) procedures in general use in our laboratory, using appropriate standards for human peptides. The gastrin RIA employs an antiserum that recognizes the gastrin COOH-terminus, but does not detect the deamidated gastrin at molar concentrations 1000-fold higher than the corresponding amidated peptide [13]. One pancreas weighing 6.4 g was extracted in 5 vol. of acid-alcohol (0.2 M HC1/75 ~o ethanol) with a Teflon tissue grinder. The acid-alcohol extract was precipitated with 8 vol. of acetone at - 30 °C overnight. The precipitate was collected by centrifugation and the supernatant was discarded. The precipitate was dissolved in 8.5 ml of 1 M acetic acid and contained 309 nmol insulin, 148 nmol glucagon and 40 nmol PP. A C18 Sep-Pak cartridge (Water Associates, Milford, MA) was prepared by washing with 10 ml ethanol and 10 mt distilled water. The redissolved precipitate was then pumped through at 1 ml/min. The cartridge was washed with 6 ml 0.1 ~o trifluoroacetic acid (TFA) and eluted with 2 ml 0.1 ~o TFA/60~o acetonitrile (ACN). The eluate was further purified by HPLC. The eluate was applied to a MB C~8 radial-pak column (Waters Associates) and eluted with a linear gradient from 20-60~o ACN in 0.13~o heptafluorobutyric acid (HFBA). Following this step, the major peaks of insulin and PP were pure, but the glucagon was contaminated with insulin. The insulin was reduced and alkylated, and the A and B chains were separated by a single HPLC step using a Nova C 18 column which was eluted with a linear gradient from 0-60 ~o ACN in 0.1 ~o TFA. The MB C~8 peak containing glucagon and small amounts of insulin was reduced and alkylated. The glucagon was then separated from the A and B insulin chains by an HPLC step using a Nova C~8 column. For the purificiation of VIP the colons from four monkeys were thawed prior to removal of the mucosa by scraping. The remaining muscle (183 g total) was extracted with 5 vol. 0.1 M HC1/90~o ethanol. The acid-alcohol extract was precipitated with 3 vol. of acetone and stored overnight at - 30 °C. The precipitate that formed was
41 removed by centrifugation. Following centrifugation the supernatant was discarded. The precipitate was redissolved in 20 ml acetic acid and contained 10.5 nmol VIP. The acetic acid solution was concentrated on a C~8 Sep-Pak cartridge, which was eluted with 2 ml 0.1 ~o TFA/60 ~o ACN. The eluate containing 7.6 nmol VIP was further processed by dilution with 8 ml 0.05 M Na acetate (pH 5) and passed through a CM Sep-Pak cartridge. The adsorbed VIP was eluted with 5 ml 0.05 M Na acetate/0.5 M NaC1 (7.1 nmol). Final purification was effected by four HPLC steps. For the purificiation of gastrin the stomachs of eight monkeys were defrosted in methanol and the antral mucosa separated. The mucosa (43 g) was boiled in H20 for 5 min, cooled and then extracted in 10 vol. of 0.1 M N H n C O 3 (22 nmol). The suspension was centrifuged at 3000g for 30 min at 4°C. The supernatant was further clarified by the addition of 2 vol. of acetone and stored overnight at - 30 ° C. The precipitate that formed was removed by centrifugation. The extract was pumped through two QMA cartridges (Waters Associates) connected in series. Each cartridge was washed with 10 ml 0.05 M Tris (pH 7) (Tris) and eluted with 4 ml Tris/1 M NaC1. The combined eluates containing 14 nmol gastrin were applied to a Sephadex G50SF (450 ml) column which was eluted at a flow rate of 60 ml/h with Tris. The elution fractions containing the major peaks of gastrin were pooled, applied to a MB C18 radial-pak column and eluted with a linear gradient from 0-30~o ACN in Tris. The NH2-terminus of the major peak was enzymatically deblocked with pyroglutamyl aminopeptidase (PAP, Sigma Chemical Co.) at a ratio of 2 units PAP/nmol 'little' gastrin in a volume of 200/~1. Deblocked peptides were repurified on a Nova C~8 column prior to sequence analysis. The COOH-terminal sequence of monkey gastrin was obtained by digesting intact gastrin (0.4 nmol) with 1 #g endoproteinase Glu-C (Boehringer-Mannheim Biochemicals, Indianapolis, IN) in 0.1 M N H n C O 3 overnight at room temperature. The mixture was sequenced directly. The purified peptides (50-200 pmol) were sequenced on an automated gas-phase sequencer with an on-line PTH amino acid analyzer (Applied Biosystems, Foster City, CA).
Results
The HPLC steps in the purification of the pancreatic peptides are shown in Fig. 1. Following a single step (left) the insulin peak and the PP peak were sufficiently pure for sequencing, but the glucagon peak contained a minor insulin contamination. The insulin from the peak fraction was reduced and alkylated, and the pure A and B chains were separated by H P L C in preparation for sequence analysis (Fig. 1, top right). The A and B chains were completely sequenced and shown to be identical to the A and B chains of human insulin. Following reduction and alkylation of the insulin-contaminated glucagon peak the glucagon was separated from the contaminating A and B chains by HPLC. (Fig. 1, lower right). Direct sequencing provided the sequence of the 24 N-terminal amino acids. Following treatment with endoproteinase Arg-C, the overlapping C-terminus fragment was sequenced. The monkey glucagon was identical to the usual mammalian glucagon. The PP was completely sequenced and shown to be identical to the corresponding human peptide.
42 MB CI8
....
05
,Nqm°l~;t R~/o ql 2t7 • 72 72 3; B 26,6 76 ,
'
Nova CI8
In~m 302 Gklcogon IOO
A
.I1/./"/1
Chmn
60
.11 ././
