Isolation and characterization of rabbit gastrin

Pepttdes, Vol 9, pp 763-769 ©PergamonPress plc, 1988 Pnnted m the U S A

0196-9781/88$3 00 + 00

Isolation and Characterization of Rabbit Gastrin R O U L A N J I A N G , * V E R E N A D. H U E B N E R , : ~ T E R R Y D. LEE,~: P E T E R C H E W , t F. J. H O , t J O H N E. S H I V E L Y , ~ J O H N H. W A L S H ' ~ A N D J O S E P H R. R E E V E , J R . t ~

*The First Teaching Hospital of China Medical University, Peoples Republic" of China tCenter Jor Ulcer Research and Education, V.A. Wadsworth, Department of Medicme UCLA School of Medicme, Los Angeles, CA 90073 5;Division oJ Immunology, Beckman Research Institute of City oJ Hope, Duarte, CA 91010 R e c e i v e d 17 A u g u s t 1987 JIANG, R , V D HUEBNER, T D. LEE, P CHEW, F J. HO, J E SHIVELY, J H WALSH AND J R REEVE, JR.

lsolatton and ~haractertzatton oJ rabbtt gastrm PEPTIDES 9(4) 763-769, 1988 --The heptadecapeptlde form of rabbit gastrm was extracted from 16 rabbit antra and purified by a combination of DEAE Sephadex, C-18 SEP PAK cartridges, fast performance liqmd chromatography (FPLC) and reverse phase high pressure hqmd chromatography (HPLC) steps After the HPLC punficatlon, a sharp, single peak of gastrm-hke lmmunoreactlvlty was detected that had the same absorption to lmmunoreactlvity ratio as human gastrm An amino terminal pyrrohdone carboxyhc acid blocking group was removed by incubation with pyrrohdone carboxyhc peptldase The amino acid analysis, mlcrosequence analys~s and mass spectrometry all confirmed the structure of rabbit gastrm being pQGPWLQEEEEAYGWMDFamlde This sequence is identical to human gastrln-17 except for glutamlne in posJtlon 6 which replaces glutamate m human gastrm Both sulfated and unsulfated rabbit gastrln-17 were characterized by mass spectrometry Gastrm

Rabbit

Fast performance hqmd chromatography

NEAR the turn of the century Edklns proposed an antral substance that caused acid secretion [6]. This substance was thought to be histamine until 1938 when Komarov demonstrated that it could be precipitated wlth trifluoroacetlc acid [16]. Th~s work was continued by several investigators in the forties and f'fftles [15, 18, 24]. The first gastrm peptide was ~solated and characterized m the early Slxtles from porcine antral extracts by Gregory and Tracy [9]. Their careful chemical analysis showed that the peptide was 17 amino acids long, it was blocked at its amino terminus by pyrrolidone carboxylic acid, that its tyrosine could be sulfated or nonsulfated, and that its carboxyl terminus was amidated. Subsequent studies have shown that several biologically active gastrins can be extracted from porcine antrum as peptides containing 6 [8], 14 [13], or 34 [10] amino acids. Amure [3] estimated antral gastrin concentrations by bioassay in several mammahan species. He demonstrated that concentrations were higher in three herbivorous species (goat, rabbit and cattle) than those from nonherbivorous species (cat, dog, pig and man). Rabbit gastrln was estimated to be 21 /zg/g of antral mucosa.

Reverse phase high pressure hquld chromatography

Rat gastrin varies from other mammahan gastrlns, hawng three differences in the first five amino acids from human gastrin [20]. One of these differences is an arginine in position 2 instead of the glycine found in other mammahan gastrlns. This change could insert a processing site not found in other gastrins, or it could slow the action of pyrrohdone carboxyhc acid peptldase. Guinea pig gastrln, another unusual mammahan gastnn, ~s one amino acxd residue shorter than the typical heptadecapeptlde [5] The absent residue represents a deletion within the five consecutwe glutamate residues typically found in mammahan gastrlns (positions 6-10) The purpose of these studxes was to determme the structure of rabb~t gastnn to permit further analysis of posttranslational and postsecretory processing m this species m comparison to metabolism of gastrin in other mammalian species. METHOD

Radiolmmunoassay of Rabbit Gastrm All purification steps used a gastrln specific radioimmunoassay based on antiserum 1611 [25]. This radioim-

1Requests for repnnts should be addressed to Dr. Joseph R. Reeve, Jr., CURE, Bldg 115, Room 115, VA Wadsworth, Los Angeles, CA 90073

763

JIANG ET AL.

764 munoassay crossreacts with CCK peptides less than 0.6% as well as with gastrm peptides

TABLE 1 RECOVERY OF IMMUNOREACTIVEPURIFICATION

Extra~ tton and Punfi~ atton Sixteen New Zealand white rabbits were killed by an overdose of barbital, their stomachs were quickly removed, opened and rinsed with cold water Antra were dissected from the stomach and put onto dry ice ~mmedmtely, then stored at -70°C until extraction. Two batches containing 44 and 52 g of tissue were boded for 10 minutes in 450 and 520 ml of 1% ammonium bicarbonate containing 0.012 M EDTA and 1 mM mercaptoethanol The t~ssues were homogenized then centrifuged at 2000xg for half an hour at 4°C. The clear supernatants were combined and applied to a DEAE Sephadex A-25 column (5 x60 cm) previously eqmhbrated with 0.1% ammonium bicarbonate, then eluted with a linear gradient to 1 6 M ammonium bicarbonate. The eluted fractions (10 ml) were monitored for gastrm immunoreactlvity using antiserum 1611 [7] and for absorbance at 280 nm. Fractions containing gastrin lmmunoreactivity were pooled and applied to four C-18 Sep Pak cartridges linked in series. This series of cartridges had been previously nnsed with 5 ml 100% acetomtrde, followed by equilibration with 20 ml of 0.1 M ammonium bicarbonate. The cartridges loaded with the DEAE purified gastrm were rinsed with 50 ml of 0.1% ammonium bicarbonate then eluted stepwise by 20 ml portions of acetonitrile (10%, 15%, 20%, 25%, 30%, 35% acetonitrlle in 0.1% ammonium bicarbonate). The Sep Pak eluates with high gastrin lmmunoreactlvity were pooled, diluted with the same volume of 0.1 M ammonium bicarbonate One-third and two-third portions were apphed separately to an FPLC mono Q anion exchange column (Pharmacia Free Chemicals) which was previously equilibrated with 0.1% ammonmm bicarbonate containing 10% acetonitrile. After loading and rinsing with the equihbration buffer the column was eluted with a linear gradient to 1 M NaCI in 0.1% ammonium bicarbonate containing 10% acetomtrile. Four tubes with high gastrin lmmunoreactlvlty were pooled and apphed onto a reverse phase C-8 pH stable HPLC column (Vydac, 4.6x250 mm). The column was equilibrated in 0.1% ammonium bicarbonate and eluted with a gradient to 50% acetonitrlle in 0.1% ammonium bicarbonate. The single absorbance peak with gastrin lmmunoreactwlty was used for characterization of rabbit gastrin.

A m m o A~ td Analyst,~ Approximately 2 nmol of rabbit gastrm from the final HPLC was put into a hydrolysis tube and dried in a vacuum desiccator over phosphorus pentoxide. After adding 0.2 ml of 6 N HC1 the tube was sealed under a vacuum, heated at 100°C for 20 hours, then opened and the HC1 removed by lyophillzation. The sample was dissolved in Beckman dilution buffer and applied to a Beckman 6300 amino acid analyzer. Ninhydrin derivatized amino acids were detected at 570 and 440 nm and the peaks were integrated with a Nelson Analytical data system.

Removal o f the Pyrrohdone Carboxylic Ac ld ResMue Sequence determination by the Edman degradation has been prevented by the presence of pyrrohdone carboxylic acid (PCA) at the amino terminus of every mammalian heptadecagastrin so far characterized. Because of the similar amino acid composltmn of rabbit gastnn to human gastrin, it

Step Extract DEAE Sep Pakt FPLC:~ HPLC§

lmmunoreactlve Gastrln (nmole)

Step Recovery (%)

Cumulative Recovery* (%)

88 74 30 25 19

-84 61 89 86

100 84 34 28 22

*Cumulative recoveries are not the product of step recoveries since at most steps all of the recovered lmmunoreactlvlty was not used m the following step +Only 49 of the 74 nmoles recovered from the DEAE column was used for SepPak step. :~Only 28 of the 30 nmoles recovered trom the SepPak were used for the FPLC step §Only 22 of the 26 nmoles recovered from the FPLC step was used for the HPLC step

was expected that a PCA group would also have to be removed from rabbit gastrm before its sequence analysis. Digestions with pyroglutamate amino peptldase (PCA peptldase) were done using two sources of enzyme and two shghtly different methods. Both methods were adapted from those used by Podell [19]. In the first method, 1 nmole of HPLC purified rabbit gastrin was digested in a l ml reaction volume containing deblocking buffer [0.1 M monosodium phosphate pH 8, 5 mM in dithiothreitol (DTT), 10 mM in disodium EDTA, 5%. in glycerin (v/v)]. A 500-fold excess of PCA peptidase (1.1 mg, Sigma from bovine liver) was added and the tube was flushed with nitrogen, mixed and sealed. The reaction was incubated at 4°C for 2 hours. An additional 1.1 mg of enzyme was then added and the mixture was incubated at room temperature for 2 hours. The unblocked gastrin was separated from the unreacted gastrin by HPLC and used for mlcrosequence analysis As judged by the HPLC immunoreactivity and absorbance profile, only 25% of pyrrolidone carboxylic acid groups were removed from the amino terminus of gastrin. The second PCA peptldase reaction contained 1.5 nmol of purified rabbit gastrin in 0.2 ml of the deblocking buffer. A 20-fold excess of Boehringer Mannheim PCA peptldase (0.062 mg, from bovine liver) was added and the mixture flushed with nitrogen and the reaction incubated at 4°C for 16 hours. The unblocked gastrin (30% of the total) was separated from the unreacted gastrln by HPLC and used for mlcrosequence analysis.

Mass Spectral Analysts Samples for FAB mass spectrometry need to be completely free of salt before their analysis. Although ammonium bicarbonate is volatile, there ~s sufficient salt present after evaporation to prevent a mass spectrum from being obtained. Therefore, prior to analysis by mass spectrometry, samples were desalted by reverse phase chromatography on a Vydac pH stable C-8 column (4.9 m m x 2 5 cm), Elution buffers were (A) 0.1% trlethylamine-acetate pH 8.6, (B) 50%

RABBIT GASTRIN STRUCTURE

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FIG. 1 Punficatton of rabbit gastrin. (A) Amon-exchange chromatography of a 1% ammonium bicarbonate (containing 0 01% EDTA and 1 mM mercaptoethanol) extract of rabbit antrum. The extract was apphed onto a DEAE Sephadex A25 column (5×60 cm) previously equilibrated with 0.1% ammonmm bicarbonate, then eluted with a gradwnt to 1 6 M ammomum b]carbonate The elutlon profile is shown for conductivity (---), absorbance at 280 nm (--), and gastnn-hke lmmunoreactmvity as measured by anUbody 1611 (O). (B) Eluted fractions with high lmmunoreactive gastnn-hke peptlde were pooled and loaded on C18 Sep Pak cartridges, previously equihbrated with 100% acetonltrlle, followed by 0.1 M ammomum bicarbonate, then eluted with a gradient to increasing percentage of acetonitrlle The elutlon profile for absorbance at 280 nm (---) and gastnn-hke lmmunoreactlvlty as measured by antiserum 1611 (--) are shown. (C) Gradient FPLC purification or rabbit gastrm-hke lmmunoreactlvlty previously purified by DEAE Sephadex A25 column and C18 Sep Pak cartridge The material elutmg with 20% acetonltrlle and 25% acetomtnle was pooled and applied to a Mono Q FPLC column equihbrated with 0.1% ammonium bicarbonate and 10% acetomtrile, then eluted with hnear gradient to 1 M NaCI m the same buffer over 200 minutes The elutlon profile for absorbance at 220 nm and 280 nm (--) for the gradient of NaC1 (---), and for the gastrln-hke lmmunoreactlvlty as measured by antibody 1611 (open bars) are shown. The gastrm-hke peptide pooled for the HPLC Is shown by the solid bar (11). (D) Gradient HPLC purification of rabbit gastrln-like lmmunoreactlvity previously purified by anion exchange chromatography C18 Sep Pak cartridges, and FPLC The C8 HPLC column was equilibrated m 0.1% amino bicarbonate (buffer A), then eluted with a linear gradient to 50% acetomtnle/50% buffer A The elution profile for absorbance at 220 nm (--), % acetonitnle (---).

766

J I A N G ET A L

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Rabbit Gastnn

Human Gastnn

0 002-

Rabb~t + Human Gastnn

~ ¢-

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TABLE 2 THE AMINO ACID COMPOSITION OF RABBIT AND HUMAN GASTR1N (BEFORE TREATMENT WITH PCA PEPTIDASE) Rabb~t pmole/ pmole PeptJde

Human pmole/ pmole PeptMe

Expected Human Composmon

15 04 08 56 10 29 1I 0 I 08 0 1 12 08 09 0 1 01 01 nd

18 05 12 58 10 35 I I 0 1 07 0 1 I2 09 09 0 I 0 I 01 nd

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FIG 2 Isocratlc HPLC elutlon profiles of rabNt gastrm and human gastrm m 15% acetonltrfle.

acetonltrile in buffer A. The elutlons were m o n i t o r e d by abs o r b a n c e at 280 nm. A p p r o x i m a t e l y 1 nmole samples of rabbit gastrin were analyzed on with J E O L HX100 H F mass s p e c t r o m e t e r operating at 5 kV accelerating potential, 3000 resolution, and equipped with a 3 kV xenon atom source [ 17]

Asp Thr Ser Glu Pro Gly Ala Val Met lie Leu Tyr Phe His Lys Arg Trp

Mt¢ ro~equen~ e Analysts U n b l o c k e d rabbit gastrins (25% of the purified material after p y r r o h d o n e c a r b o x y h c acid residues were r e m o v e d ) w e r e subjected to a u t o m a t e d E d m a n degradations on a modified B e c k m a n 890C s e q u e n c e r as previously described [22]. The P T H amino acid derivative were identified by H P L C as previously described [14] RESULTS Extra~ non and Purifi~ atton R e c o v e r y of gastrln lmmunoreactlvlty for each punficatlon step is shown in Table 1. The c o m b i n e d a m o u n t of gastrln i m m u n o r e a c t i v i t y in the two crude extracts was 87.8 nmol. The elution profile of gastrin-hke i m m u n o r e a c t w i t y from the D E A E column is s h o w n in Fig. IA Two-thirds of the gastrin i m m u n o r e a c t i v i t y (49 nmole) eluted away from the majority of the a b s o r b a n c e at 280 nm and was used for further Sep Pak purification. Figure 1 B shows that the majority of the gastrln l m m u n o r e a c t l v l t y (23.3 nmol) eluted in 2(F/b acetonltrile while the majority of a b s o r b a n c e at 280 nm eluted in 10% acetonitnle. The l m m u n o r e a c t i v l t y in 20% and 25% acetonitrlle was pooled, diluted and loaded on the F P L C c o l u m n in two batches containing one-third and two-thirds of the pooled immunoreactlvity. The F P L C elution profile for one-third (10 nmole) of the Sep Pak purified gastrln immunoreactivity is shown in Fig. IC. O v e r 80% of the immunoreactivity eluted in a peak corresponding to an o b s e r v a b l e abs o r b a n c e at 220 nm peak The other two-thirds of the Sep Pak purified material was c h r o m a t o g r a p h e d on the F P L C and the i m m u n o r e a c t i v i t y eluting in the same region of both c o l u m n s was pooled (23 nmole) and used for H P L C purification. A minor peak o f gastrm i m m u n o r e a c t i v l t y which eluted before the major peak was also pooled, and purified by C-8 r e v e r s e phase H P L C and c h a r a c t e r i z e d by mass spectral

analysis (purification data not shown). Figure ID shows the C-8 reverse phase eluUon profile of a portion (7.7) nmole of the major peak of rabbit gastrin i m m u n o r e a c t i v i t y previously purified by F P L C It eluted in the same region as synthet,c human gastnn-17I and had the same l m m u n o r e a c t l v e to absorbance ratio as human gastrm-171 c h r o m a t o g r a p h e d in the same m a n n e r lso¢ rattc Comparison oJ Rabbit and Human Gastrms Rabbit and human gastrln eluted apparently in the same position during gradient elution (Fig 1D). F o r more careful c o m p a r i s o n we determined conditions of Isocratlc elution of human gastrln on the C-8 pH stable column. U n d e r these conditions rabbit gastrin eluted at 6.5 min, while human gastrin eluted at 7.5 mln. W h e n approximately equal amounts of the two peptldes w e r e colnjected, rabbit gastrln eluted at 7.0 and human gastrln at 7.7 mln (Fig. 2), clearly demonstrating different retention times A m m o Actd Analysis Before treatment by P C A peptidase, 1.7 nmol of the purified rabbit gastrln from the C-8 H P L C step was analyzed. F o r c o m p a r i s o n a similar a m o u n t of synthetic human gastrin-I was c h r o m a t o g r a p h e d on the same c o l u m n and analyzed (Table 2). The results indicated that the composition of rabbit gastnn is identical to the human hept a d e c a p e p t i d e gastrin Both amino acid analyses show threonine, serlne and glycine residues which w e r e not found in amino acid sequences. T h e s e extra residues represent contamination that could have c o m e f r o m H P L C buffers, glass tubes used for collecting fractions, or glass hydrolysis

RABBIT GASTRIN STRUCTURE

767

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FIG. 3 Negative ion FAB mass spectrum of approximately 1 nmole of rabbit gastrm G-17. Spectrum zs the average of four scans and normahzed to the most abundant ion m the molecular ion cluster (m/z 2096) (A) Region of the spectrum showing the deprotonated molecular 1on calculated from the expected elemental composition (B) Region of the spectrum from m/z 950 to 2350 showing the singly (m/z 2096) and doubly (m/z 1407) charged molecular 1on clusters Fragment ions observed are consistent w~th the expected sequence

tubes. The fact that the synthetic peptlde contained more of these residues than the natural rabbit gastrm supports the conclusion that it was actually contamination.

Removal oJ Pyrrohdone Carboxyhc Actd As described in the Method section, two different methods of pyrrohdone carboxyhc removal were utilized. The first removed the first three residues from rabbit gastrin (see below), probably reflecting contamination of the PCA preparation by another peptidase actwlty. The second method removed only the amino terminal pyrrohdone carboxyhc acid.

Mass Spectral Analysts Mass spectral analysis of rabbit gastnn m the negative ion mode gave a molecular ion (M-H) of 2174.9 (not shown). The molecular weight of rabbit gastnn is therefore 2175.9 mass units. A less predominant peak of gastrm immunoreactivity

eluted earher from the mono Q FPLC column. It also was purified by reverse phase and prepared for analysis by HPLC The molecular weight obtained for this form was 2095.8 mass units (Fig. 3). These results are consistent with the later eluting gastrin having the higher molecular weight due to a sulfated tyroslne. The weight of the unsulfated form was one mass unit less than synthetic human gastnn which is consistent with glutamlne being present in position 6 of rabbit gastrin instead of the glutamate that is present in human gastrln.

Mtcrosequence Analysts Microsequence analysis of rabbit gastrln which had been digested with PCA peptidase using the first set of conditions gave the sequence W L Q E E E E A Y G W M D (Table 3) This corresponds to a heptadecapeptlde with the three amino terminal amino acids deleted. The carboxyl terminal phenylalanine present in the amino acid analysis was not detected but phenylalanme-amlde IS easily extracted from

768

JIANG ET AL. TABLE 3

Rabbit

MICROSEQUENCE ANALYSIS OF UNBLOCKED RABBIT GASTR1N

Digest No 1 Cycle

Amino Acid

Digest No 2

pmoles

Cycle 1

1 2 3 4 5 6 7 8 9 10 11 12 13

Trp *Lys/Leu Gin Glu Glu Glu Glu Ala Tyr Gly Trp Met Asp

26 23 9 14 18 13 16 18 12 9 5 9 2

2 3 4 5 6 7 8 9 10 I1 12

Amino Acid

pmoles

Gly Pro Trp Leu Gin Glu Glu Glu Glu Ala Tyr Gly

54 23 18 38 25 25 29 29 27 12 11 5

*Could not be dlstmgmshed Lys and Leu were not resolved under the conditions for PTH-ammo aod chromatography

the reaction c h a m b e r by s e q u e n c e analysis buffers [21] M1c r o s e q u e n c e analysis of rabbit g a s t n n which had been digested by the second P C A peptmdase m e t h o d gave the seq u e n c e G P W L Q E E E A Y G (Table 3). These overlapping seq u e n c e s , c o m b i n e d with amino acid analysis and mass spectral analysis, permit the conclusion that the structure of rabbit gastrm Js p Q G P W L Q E E E E A Y G W M D F a m l d e DISCUSSION The structure of several mammalian gastrlns has been determined including, rat [20], guinea pig [5], cat [2], dog [4,12], pig [8--10, 13], c o w , [1], sheep [1], g o a t [4] and man [11] (Fig. 4). Rat varies the m o s t f r o m o t h e r species It is the only species to have an arglnlne in position 2 and a proline in position 4. The fifth p o s m o n of gastrm varies the most, being o c c u p i e d by m e t h l o n m e , alanlne, leucine, or valme in various species. The o t h e r residue that differs in m a n y species Is p o s m o n 8, which is e~ther glutamate or alanlne m various m a m m a l s . Figure 4 shows the most recent report [4] on the structure of dog gastrln containing alanlne in position 10 (alanine is also in position 10 in o t h e r mammals) although an earlier report on dog gastrin s h o w e d the alanme to be m p o s l u o n 8 [12]. B o t h sulfated and nonsulfated heptadecapepttde rabbit gastrIn w e r e c h a r a c t e r i z e d by mass spectral analysis. M o r e than four times m o r e sulfated rabbit gastrin than nonsulfated gastrIn was obtained d u n n g the final p u n f i c a t i o n step. This may or m a y not reflect the concentrations stored m the

pQ G P W L Q E E E E A Y G W M D F #

Human

E

Pig

M E

Goat

V E

A

Cow

V E

A

Sheep

V E

A

Dog

M E

A

Cat

M E

A

Guinea Rat

pig

A E R

*

A

P M E

FIG 4. Comparison of the structures of mammahan gastrms The structure of rabbit gastrln is shown at the top using the one letter code The pQ at the amino terminus indicates that pyrrohdone carboxyhc acid is the amino terminus of rabbit gastnn The positions where other mammalian gastrms are different from rabbit gastrln are shown below using the one letter code for the amino acids. The * symbol indicates that guinea pig gastnn is lacking one of the glutamlc acid residues found in other mammalian gastrms The # symbol indicates that the carboxyl terminus of all mammalian gastrms is phenylalanme-amide

antrum since the two previous steps may h a v e eluted sulfated and nonsulfated gastnns differently. Rabbit gastrin-34 was not d e t e c t e d d u n n g these studies, but it is likely that it eluted earlier on the first D E A E column than the majority of the gastrm l m m u n o r e a c t w i t y and was not pooled for further purification. Rabbit gastrin is nearly idenUcal to human gastrm (Fig 4). In the two species, gastnn varies only in position 6, at which the rabbit peptlde has glutamlne and the human has glutamate. All o t h e r m a m m a l i a n gastrlns have glutamate in this position. Pig, cat and dog have m e t h l o n m e in posxtxon 5, while human and rabbit have leucme in this position. In these species, but not m rabbit o r man, methlonlne oxidation to m e t h l o n m e sulfone at position 5 could alter the metabolism of gastrln. The entire range of physiological activities of gastrm are possessed by the C-terminal tetrapeptlde s e q u e n c e TyrMet-Asp-Phe-NH,, [23]. Therefore, rabbit gastrln should have nearly identical biological effects to other mammalian gastnns Its similar biological actw~ty and close structural h o m o l o g y to human g a s t n n make rabbit an ideal species for studying the p o s t s e c r e t o r y processing of e n d o g e n o u s l y released gastnn.

ACKNOWLEDGEMENTS This work was supported by National Institute of Health Grants DK 331155 and DK 17328 and the Veterans Admlmstratlon

REFERENCES

1. Agarwal, K L., J. Beacham, P H. Bentley, R A Gregory, G W. Kenner, R C Sheppard and H J Tracy Isolation, structure and synthesis of ovlne and bowne gastrlns Nature 219: 614-615, 1968

2 Agarwal, K L., G W Kenner and R C Sheppard Fehne gastnn An example of pephde sequence analysis by mass spectrometry J A m Chem So~ 91: 3096-3097, 1969

RABBIT GASTRIN STRUCTURE

3. Amure, B O. and A. Omole Comparative study of antral gastrln activity m some mammals. Br J Pharmacol 41: 62%639, 1971 4. Bonato, C., J. Eng, J D Hulmes, M Mledel, Y. C Pan and R S Yalow. Sequences o f g a s m n s purified from a single antrum of dog and of goat Pepttdes 7: 68%693, 1986 5 Bonato, C., J Eng, Y C. Pan, M Chang, J. D Hulmes and R S Yalow Guinea pig " h t t l e " gastrln is a hexadecapeptide Ltje Scl 37: 2563-2568, 1985 6. Edkms, J. S On the chemical mechamsm of gastric secretion. Proc R Soc Lond [Blol] 76: 376, 1905 7 Eysselem, V E , V Maxwell, T Reedy, E Wunsch and J H Walsh Similar acid stlmulatory potenoes of synthetic human big and httle gastrins m man, J Chn Invest 73: 1284--1290, 1984. 8 Gregory, R A., G J Dockray, J R Reeve, J r , J E Shlvely and C. Miller Isolation from porcine antral mucosa of a hexapeptlde corresponding to the C-terminal sequence of gastrin Peptldes 4: 31%323, 1983. 9. Gregory, R. A and H J. Tracy The const~tutmn and properties of two gastnns extracted from hog antral mucosa Part 1 The isolation of two gastrlns from hog antral mucosa Part ll The properties of two gastrlns from hog antral muscosa Gut 5: 103117, 1964 10. Gregory, R A and H J Tracy The chemistry of the gastrlns. some recent advances In GastromtesttnaIHormones A Syrnpowum, edited by J C Thompson Austin. Umverslty of Texas Press, 1975, pp 13-24 11. Gregory, R. A , H. J Tracy and M 1 Grossman Isolation of two gastnns from human antral muscosa Nature 209: 583, 1966, 12 Gregory, R A., H J Tracy, M l Grossman, D De Valolsand R Llchter Isolation of canine gastrln Expertentm 25: 345-346, 1969. 13 Gregory, R. A , H J Tracy, J 1 H a m s , M J Runswlck, S Moore, G W. Kenner and R Ramage Mlnlgastrln, corrected structure and synthesis Hoppe Seylers Z Physiol Comp 360: 73-80, 1979 14 Hawke, D , P M Yuan and J E Shwely Mlcrosequence analysis of peptldes and proteins 11 Separation of amino acid phenylthlohydantoln derivatives by high-performance liquid chromatography on octadecylsllane supports. Anal Blo~hem 120: 302-311, 1982

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