A new molecular form of PYY: Structural characterization of human PYY(3–36) and PYY(1–36)

Peptides,Vol. 10, pp. 797-803. ©PergamonPress plc. 1989. Printedin the U.S.A.

0196-9781/89$3.00 + .00

A New Molecular Form of PYY: Structural Characterization of Human PYY(3-36) and PYY(1-36) G E R T A. E B E R L E I N , .1 V I K T O R E. E Y S S E L E I N , t M A ' I q ' H I A S SCHAEFFER,~: P E T E R LAYER,~: D A N I E L GRANDT,~t H A R A L D G O E B E L L , ~ W O L F G A N G NIEBEL,~t M I C H A E L D A V I S , § T E R R Y D. LEE,§ J O H N E. S H I V E L Y § A N D J O S E P H R. R E E V E , JR.¶

*California Biotechnology Inc., Mountain View, CA 94043 "PHarbor-UCLA Medical Center, Department of Gastroenterology Center for Inflammatory Bowel Disease, Torrance, CA 90509 SUniversity of Essen, Department of Gastroenterology and Surgery, D-4300 Essen I,FRG §Division of lmmunology, City of Hope Research Institute, Duarte, CA 91010 ¶Center for Ulcer Research and Education, VA Wadsworth Medical Center Department of Medicine, UCLA, Los Angeles, CA 90073 Received 17 February 1989

EBERLEIN, G. A., V. E. EYSSELEIN, M. SCHAEFFER, P. LAYER, D. GRANDT, H. GOEBELL, W. NIEBEL, M. DAVIS, T. D. LEE, J. E. SHIVELY AND J. R. REEVE, JR. A new molecularform of PYY:Structuralcharacterizationof human PYY(3-36) and PYY(1-36). PEPTIDES 10(4) 797-803, 1989.--A radioimmunoassay was developed using an antibody raised in rabbits against synthetic porcine PYY. This radioimmunoassay was used to detect PYY immunoreactivity in human intestinal extracts. Human colonic mucosa was extracted with acid, centrifuged and the supematant concentrated by low pressure preparative reverse phase chromatography. A subsequent C-18 reverse phase HPLC step separated two peaks of PYY immunoreactivity. Each peak was purified by sequential steps of ion-exchange FPLC and reverse phase HPLC. In the final purification step single absorbance peaks were associated with PYY immunoreactivity. Microsequence, amino acid, and mass spectral analysis of the intact and tryptic fragments of the two popfides were consistent with the structures: YPIKPF-.AIKiEDASPEELNRYYASLRHYLNLVTRQRY-amide[human PYY(1-36)] and --IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY-amide [human PYY(3-36)]. Human PYY(I-36) differs from porcine PYY only at position 3, with lie instead of Ala, and position 18, with Ash instead of Ser. PYY(3-36) may differ in its biological activity from the intact peptide. Its high proportions in the colon suggest that it is released into the circulation where it could act as a partial antagonist of PYY(I-36). Peptide YY (PYY) Neuropeptide Y (NPY) Gastrointestinal hormones Pancreatic secretion Gastric acid secretion Motility Microsequence analysis High performance liquid chromatography Fast protein liquid chromatography Molecular heterogeneity Radioimmunoassay SINCE so many bioactive peptides contain a carboxyl terminal amide, Tatemoto and Mutt have developed a method to purify peptides by detection of these groups (28). Using this chemical detection method, a peptide containing carboxyl terminal tyrosine amide was purified from porcine intestinal extracts. This peptide, of 36 amino acids with an amino terminal tyrosine and a carboxyl terminal tyrosine-amide, was named peptide YY (PYY) (29). The structure of rat PYY was recently shown to be identical to porcine PYY (I0). The structure of human PYY(l-36) was concurrently presented by Tatemoto (31) and our laboratories (12) at the Seventh

International Symposium on Gastrointestinal Hormones. Tatemoto's group recently reported PYY(I-36) as the only form detected following his purification procedure (32). However, more than one form has been detected during reverse phase HPLC of colon extracts. Roddy et al. (26) detected two peaks of routine PYY immunoreactivity from colonic extracts with one particular antibody (Ah 80), but only one peak with another antibody (Ab 213), suggesting that more than one molecular form of PYY was present in these colonic extracts. These same two antibodies detected in human colonic extracts single major immunoreactive peaks eluting approximately 2 rain differently, suggesting the existence of two

tRequests for reprints should be addressed to C.rertA. Eberlein, California Biotechnology Inc., 2450 Bayshore Parkway, Mountain View, CA 94043.

797

EBERLEIN ET AL

798

1 O0

TABLE 1

pyy

RECOVERY OF IMMUNOREACTIVE HUMAN" PYY DURING PURIFICATION

_J

90 0 I-"

Purification Step

8O

UJ t,U IZ I,,I,,

7O

NPY i

60 o

~,

i

i

g

i

i

io

1oo

i

i

Immunoreactive PYY (pmoles)

Step Recovery (%)

Colon extract (70 g tissue)

620

--

I) Preparative C-18 chromatography

480

77

PP 1ooo

pM FIG. 1. Displacementcurve for porcine peptide YY (PYY). Antibody 1201 was raised against porcinepeptide YY. There was no cross-reaction with human pancreatic polypeptide (PP) or neurotensin,gastrin G-17 I, and CCK-8. Using porcine neuropeptide Y (NPY), a cross-reactivityof 0.5% was found.

forms. Chen has detected several molecular forms of human PYY in plasma extracts (8). These papers strongly suggest the presence of multiple molecular forms of stored and circulating PYY. Porcine PYY has several biologic actions in animals, including vasoconstriction, inhibition of pancreatic and gastric secretion, and inhibition of gastrointestinal motility (1, 3, 9, 19, 21). Radioimmunoassays have been developed for porcine PYY (33). The highest concentrations of PYY have been detected in the distal small intestine and colon (2, 8, 26, 33), where it is found in endocrine cells (13,18). Because of this location, the release of PYY into the circulation after a meal, and the actions of PYY, it has been suggested that PYY is the hormone responsible for terminating upper gastrointestinal functions when nutrients reach the distal part of the intestine (22). The aim of our study was to determine the structure of new molecular forms of human PYY in order to develop accurate standards for measurement of PYY, and for testing of structurefunction relationships between human PYY(1-36) and other forms. ABBREVIATIONS PYY: peptide YY; PYY-LI: PYY-like immunoreactivity;NPY: neuropeptide Y; TFA: trifluoroacetic acid; HPLC: high peformance liquid chromatography; FPLC: fast protein liquid chromatography; RIA: radioimmunassay; DAB: 1,4-diamino-butane. METHOD

Materials The following chemicals and solvents were purchased: from E. Merck: acetonitrile, trifluoroacetic acid (I'FA), 2-mercaptoethanol; from Aldrich: 1,4-di~uninobutane (DAB); from Signm: porcine PYY and glutaraldehyde; from Waters/Millipore: C-18 Sep Pak cartridges; and from Behringwerke: incomplete and complete Freund's adjuvant.

Radioimmunoassay of PYY The antibody 1201 was raised in our laboratory as follows: Porcine PYY was coupled to bovine albumin by glutaraldehyde, emulsified with complete Freund's adjuvant, and injected intracutaneously in six rabbits. The rabbits were boosted at intervals of

2) C-18 HPLC

a: 540 b: 270

180 total*

3) FPLC

a: 400 b: 195

74 72

4) C-18 HPLC

a: 500 b: 95

125 48

5) Phenyl HPLC

a: 287 b: 145

57 152

6) CN HPLC

a: 230 b: 190

80 132

7) C-4 HPLC

a: 208"1" b: 247"I"

90 130

a, b: = PYY form A and B; *comparablerecovery for both PYY forms was assumed: tamounts calculatedby amino acid analysis.

four weeks by another injection using incomplete Freund's adjuvant. After the third immunization a titer of 1:50,000 was obtained. Porcine PYY was labeled with ~2al by chloramine T. The RIA was performed at pH 8.4 in 0.02 M veronai buffer containing 0.17% (w/v) bovine serum albumin. Immunoreactive PYY was diluted in HPLC buffer containing 2-mercaptoethanol (acetonitrile/0.1% T F A / 2 - ~ I , 32.5: 62.5:5, v/v/v). After an incubation period of 16-25 hr at 4°C, free PYY was separated from antibody-bound PYY by dextran-coated charcoal.

Extraction of PYY From Human Intestine Tissue procurement. The distal aorta of a multiorgan donor was continously perfused in situ at 4°C with Ero-collins solution (Fresenius AG, Bad Homburg, FRG). The arteria mesenterica superior and inferior and the truncus coeliacus were opened for the perfusion of the intestine. The intestine with the pancreas w a s removed within 2 hr after starting the perfusion. The colon was rinsed with cold water, boiled in water for 10 rain, and the mucosa was separated from the muscle layer. The mueosa was immediately frozen. Purification of human PYY. The mucosa (70 g) was homogenized in 140 ml 4% aqueous TFA at 4*(2. The extract w a s centrifuged for 10 minutes at 3,500xg and the pellet w a s reextracted with 140 ml of 2% aqueous TFA. The supec~,~t w a s loaded onto a preparative 25 x 50 mm low pressure reverse l)h~e C-18 column at a flow rate of 10 ml/min. The column was rinsed with 0.1% aqueous TFA (50 ml) and eluted step-wise with a mixture of acetonitrile/0.1% TFA (40:60, v/v) at a flow rate of 2 ml/min. PYY-LI eluted between 10 to 40 ml of eluate. The eb_mt_e

HUMAN PYY(3-36) AND (1-36) STRUCTURES

799

A. - 1 .O

/

20-

/ / 15-

T ¢: v

s

./

/

-40

0.10-

I

/ / /

10-

./

-0.5

T

°

/

/"

./

/

/

J

./

./ 30

T i

T

"l '

ZO

0

n* I--

N

O 0 led U

¢¢

_1

0.05-

-2O >(:L

10

~

5-

-O

OI

I

40

I

I

I

60 TIME (rain)

I

O-

-O

! 20

I

I 30

I

80

TIME

FIG. 2. Elution profile of PYY-like immunoreactivity from reverse phase HPLC. Two forms of PYY (A and B) were pooled as indicated by bars. Both forms were purified separately in the subsequent steps.

I

I 40

I

(rain)

/

B.

/ /

was diluted with 0.1% aqueous TFA (1:3, v/v) and applied to a semipreparative reverse phase C-18 HPLC column (Waters, ixBondapak, 10 micron, 7.8 ×300 mm). Immunoreactive PYY was eluted at 2 ml/min by increasing concentrations of acetonitrile: 5 minutes with 0.1% aqueous TFA, a 5-minute gradient to 20% acetonitrile, then a 70-minute gradient to 37.5% acetonitrile. The fractions containing immunoreactive PYY were pooled and applied to a cation exchange fast protein liquid chromatography (FPLC) column (LKB, TSK SP-5PW, 8 x 75 mm), which was equilibrated with 0.025 M acetate buffer containing 10% acetonitrile, pH 4.8. Immunoreactive PYY was eluted at 1 ml/min by increasing concentrations of sodium chloride: 10 minutes with acetate buffer, then a 40-minute gradient to 1 M sodium chloride in buffer. To the fractions collected (2 ml) 0.1 ml 2-mercaptoethanol and 0.10 ml 10% TFA were added to each. The immunoreactive PYY fractions were further purified by a series of reverse phase HPLC steps, including C-18, phenyl and CN columns. The purification was monitored by radioimmunoassay and absorbance at 220 and 280 nm.

- 30

/

T

./

/

0.10-

i

I

/

T

W _J

--20 ~: I-

o 0 kw U

0.05-

-10

j

A

~

O-

-0 I

20

I

!

I

4O

30 TIME

|

(rain)

Amino Acid Analysis Approximately 60 pmoles of purified peptide were lyophilized in a hydrolysis tube. The sample was hydrolysed by vapor HCI at 150°C for 20 hr. The HCI was removed and the contents dissolved in Beckman dilution buffer for amino acid analysis. The sample was applied to a Beckman 6300 amino acid analyzer.

FIG. 3. Elution profile of the PYY forms A and B in their final purifcations using a C-4 reverse phase column. These peaks were used for microsexluence and mass spectral analysis. A shows the ahsorbance profde of human PYY A [PYY(3-66)] at 220 nm. For comparison, the arrow indicates the elution position of human PYY(1-36). B shows the absorbance profile of human PYY B [PYY(I-36)] at 220 rim. For comparison, the arrow indicates the elution position of human PYY(3-36).

Tryptic Digestion and Reverse Phase HPLC Approximately 150 pmoles of intact peptide were digested with TPCK-treated trypsin (1 microgram, HPLC purified) in 0.2 ml of

0.2 M NH4HCO 3, pH 8, for 18 hr at 37°C. The tryptic digest was directly fractionated on a Vydac C-18 reverse phase column (5

EBERLEIN ET AL.

800

TABLE 2

Amino Acid I Tyrosine 2 Proline 3 Isoleucine 4 Lysine 5 Proline 6 Glut.acid 7 Alanine 8 Proline 9 Glycine 10 Glut.acid 11 Asp.acid 12 Alanine 13 Serine 14 Proline 15 Glut.acid 16 Glut.acid 17 Leucine 18 Asparagine 19 Arginine 20 Tyrosine 21 Tyrosine 22 Alanine 23 Serine 24 Leucine 25 Arginine 26 Histidine 27 Tyrosine 28 Leucine 29 Asparagine 30 Leucine 31 Valine 32 Threonine 33 Arginine 34 Glutamine 35 Arginine 36 Tyrosine

Form A PYY(3-36)

9.5 4.3 3.7 4.8 5.0 2.0 1.0 2.5 0.3 2.0 0.7 0.3 0.5 1.0 0.4 0.2 0.5 0.4 0.3 0.2 0.1

T-2

~

y r - P r o - I r e - L y e - P r o - G l u - A 1 a- l,ro-Gl y - G Iu - A s p - A " a-

SEQUENCEANALYSISOF HUMANPYY, FORM A AND B, AND THEIR TRYPTIC FRAGMENTS* Form B PYY(1-36) 2.5 0.6 1.7 0.7 0.6 1.3 1.4 0.5 0.3 0.7 1.0 0.7 <0.1 0.1 0.1 0.2 0.1

0.5 <0.1 0.2

B int. B(T-I) A

T- 1

T-2

---.~

~T-I)

---~

-.---~

--.-.~

.-...A

~

,--..~

___~

~

.,.--~

.--.A

__.A

int.

T-3 20 S e r - P r o - G i u-Gl u - L e u - A s n - A r g - T y r - T y r - A l

9.1 2.8 8.2 1.7 2.6 2.9 5.2 1.2 0.7 0.6 0.6 0.5 0.5 0.1 0.1 0.1 0.3 <0.1

11(

a-Set- Leu-

(T-~)

B int. B(T-I) B(T-2) A Int . A{T-2)

~

3O

-H i s - T y r- L e u - A s n - L e u - V a 1 - T h r - A r g - G i n - A r g - T y r - N H

1(

(r-3)

9

) [ ('-( T 4 ) '--) 1(- (T 5 ) -.~

B(T-3)

FIG. 4. Human PYY sequence analysis of the intact forms A [human PYY(3-36)] and B [human PYY(I-36)] and their tryptic fragn~nts. A int. and B int. show sequence obtained for intact form A and form B, respectively. Tryptic fragments are designated by the form they were sequenced from and their tryptic fragn~nt number [i.e., A(T-1) is form A's first tryptic fragment]. Actual yields of PTH derivatives are shown in Table 2. The arginines at the end of trypsin fragments T-2 and T-3 were determined by mass spectral analysis of these fragments. T-4 and T-5 were assigned by homology with the porcine peptide and the fact that amino acid analysis (Table 4) and mass spectral analysis (Table 3) of intact peptides are consistent with this assignment.

4.1 11 15 8.5 2.5 11 10 1

10 11 I

*Yields are calculated against 10 pmol standard. Glut.acid = glutamic acid; asp.acid = aspartic acid.

micron, 2.1 m m x 25 cm) using a 60-minute linear gradient from A (0.1% aqueous TFA) to B (0.1% aqueous TFA/acetonitrile, 1:10, v/v) at a flow rate of 0.15 ml/min.

Amino Acid Sequence Analysis Samples of purified pcptides or their purified tryptic fragments were subjected to antomat~d E d m ~ degradation on a City of Hope 925 gas phase sequencer (27), and l~aenyltlaiohydantoin derivafives of amino acids were analyzed by HPI.,C as described (14).

Mass Spectral Analysis Samples collected from reverse phase HPLC in polypropylene microoentrifuge tubes were concontrated to dryness using a vacuum centrifuge. The sample was redissolved in a few microliters of 5% (v/v) aqueous acetic acid. Approximately 0.002 ml of the

sample solution were added to 0.001 ml of the sample matrix on a 1.5 × 6 mm stainless steel sample stage. The sample matrix used was either l-thioglycerol or a mixture of dithiothreitol:dithioerythritol (5:1) (35) and camphor sulfonic acid (6 raM) (11). Positive ion spectra were obtained using a JEOL HX-100I-IF high-resolution, double-focusing, magnetic-sector mass spectrometer operating at 5 kV accelerating potential and a nominal resolution of 3000. Sample ionization was accomplished using a 6 keV Xe atom beam. A JEOL DAS000 data system was used to control instrument parameters and collect spectral data. Spectra of the intact form A and form B peptides were taken at a nominal resolution of 1000. Form B gave a weak molecular ion and the error in mass assignment (___ 1 ainu) was larger than normal (---0.3 ainu). Mass values reported for tryptic fragments are for the monoisotopic protonated molecular ion. Mass values for the intact forms A and B are the average mass of the protonated molecular ion. RESULTS

Radioimmunoassay of PYY Figure 1 shows a typical standard curve for porcine PYY. The PYY antibody 1201 was used i . a final dilution o f 1 : 5 0 , 0 0 0 . The detection limit was about 10 fmol/ml and the lDso was 50 frnol/ml. When the amounts of purified human PYY were detmrmined by measurements of absodmuce at 280 nm or amino acid analysis, the PYY antibody fully recognized human PYY. The absorbance coefficient of human PYY (7,535 M - ] c m - ~ at 280 nm) was calculated from the number of tyrosiues in the molecule. Antibody 1201 had a cross-reactivity of about 0.5% with ueuropcptide Y (NPY). The antibody did not recognize human pancreatic polypeptide (PP), neurotensin(1-13) [NT(I-13)], gastrin 17-I, cholecystokinin octapeptide (CCK-8) or the amino terminal fragment

HUMAN PYY(3-36) AND (1-36) STRUCTURES

801

TABLE 3 MASS SPECTRALANALYSISOF HUMANPYY, FORMA AND B, AND THEIRTRYI:rrlc FRAGMENTS Fragments Molecular Weight

Form A PYY(3-36)

A(T-2)

A(T-3)

Observed Calculated

4050.6 _+ 0.3 4050.5

772.2 _ 0.2 772.4

1015.6+-- 0.2 1015.6 Fragments

Form B PYY(l-36) Observed Calculated

4310 • 1" 4309.8

B(T-I)

B(T-2)

B(T-3)

2112.0 - 0.2 2112.0

772.4 --- 0.2 772.4

1015.5 -4- 0.2 1015.6

• Larger than normal error in mass accuracy due to a weak molecular ion signal.

quent chemical characterization.

TABLE 4 AMINO ACIDANALYSISOF HUMANPYY, FORMA AND B Form A PYY(3-36)

Chemical Characterization of PYY, Form A and B

Form B PYY(I-36)

Found

Expected

Found

Expected

3.0 1.0 2.2 5.3 2.8 2.4 3.1 1.0 0.8 3.6 3.0 0.9 1.8 3.6

3 1 2 5 3 1 3 1 1 4 4 1 1 4

3.0 1.0 2.3 5.5 3.4 2,6 3.2 1.1 I. I 3.9 4.4 I. 1 1.1 3.8

3 1 2 5 4 I 3 1 I 4 5 I I 4

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Histidine Lysine Arginine

1-27 of canine CCK-33 (1-27 CCK-33).

Purification of PYY Recovery of immunoreactive PYY is shown for each purification step in Table 1. During the purification procedure only two dilutions were used to detect the elution profile of human PYY. Estimates of peptide amounts therefore occasionally were made from the nonlinear portion of the standard curve which may have caused inaccuracy in determination of peptide amounts accounting for greater than 100 percent recovery. Two major forms of PYY (form A and form B) were well separated during the second preparative purification step, using a semipreparative C-18 reverse phase column (Fig. 2). The immunoreactivity in peak A was about twice that of peak B. Both forms were separately further purified by a cation exchange FPLC column and by C-18, phenyl and CN HPLC columns. Both PYY forms, A (Fig. 3A) and B (Fig. 3B), eluted as single absorbance peaks on a C-4 reverse phase HPLC column at different positions. This material was used for subse-

Sequence analysis was performed on PYY forms A and B and their purified tryptic fragments. The amino acid, microsequence, and mass spectral analyses of the intact peptide and its tryptic fragments were all consistent with form B being human PYY(136). Microsequence analysis of intact form B began with tyrosine in position 1 and continued to leucine in position 17 (Table 2, Fig. 4). Mass spectral analysis revealed a mass of 4310 daltons (Table 3), which is the expected mass for the structure shown for human PYY(I-36) (Fig. 4). Tryptic fragment T-1 of human PYY (1-36) was sequenced from the tyrosine in position 1 to asparagine in position 18, and its observed mass was that expected for human PYY(1-19). Tryptic fragment T-2 of human PYY(I-36) was sequenced from tyrosine at position 20 to leucine at position 24 (Table 2, Fig. 4), and its mass was that expected for human PYY(20-25) (Table 3). Tryptic fragment T-3 of human PYY(136) was sequenced from histidine at position 26 to threonine at position 32 (Table 2, Fig. 4) and its mass was that expected for human PYY(26--33) (Table 3). Tryptie fragments T-4 (Gln-Arg) and T-5 (Tyr-NH 2) were not recovered but the mass spectral and amino acid analysis of the intact peptide are consistent with these three amino acids being at the carboxyl terminus, and their order has been assigned by homology with porcine and rat PYY (10,29). The amino acid, microsequence, and mass spectral analyses of the intact peptide and its tryptic fragments were all consistent with form A being human PYY(3-36). Table 2, Table 3 and Fig. 4 show the data for structural assignment of the peptide. The microsequence for the intact peptide was successful through a longer region that for human PYY(1-36). This provides the necessary overlap for assignment of the order of the tryptic fragments. DISCUSSION Species differences in the structure of PYY have been suggested by radioimmunoassay results (26). Roddy et al. raised two antibodies against porcine PYY. One antibody (Ab 80) detected human and canine PYY with similar patterns of distribution in the gastrointestinal tract, whereas the other antibody (Ab 213) recognized similar amounts of PYY in canine, but not in human extracts. They concluded that there are "species related antibody recognition differences." Three potential differences in human

802

EBERLEIN E T AL.

PYY could account for its lack of recognition by antibodies raised to porcine PYY. If the antibody (213) required intact amino termini, a significant portion of the human PYY peptide [PYY(336)] would not have been detectable. The other two differences are the changes in amino acids in position 3 and 18. Position 3 of PYY is lie in humans but Ala in pigs and rat, while position 18 is Asn instead of Ser. It is important that all studies measuring human PYY have a standard containing these substitutions for validation of radioimmunoassay results. It is possible that our antibody (1201) recognizes both forms of human PYY so well because it was raised against porcine PYY coupled to bovine serum albumin through its amino terminus. This coupling could shield the amino terminus from acting as an antigen. Two forms of human PYY were detected by our antibody to porcine PYY. The two forms were first observed after separation on the C-18 reverse phase HPLC column. Both forms were purified spearately and amino acid, microsequence and mass spectral analysis of the purified peptides and its tryptic fragments allowed their structural characterization. The larger form eluting later on HPLC, form B, contained 36 amino acids, the same number detected in porcine (29), rat (10) and human PYY (12, 31, 32) whereas the smaller form, A, lacked the first two amino acids, Tyr-Pro, found at the amino terminus of human PYY(I-36). Cleavage of amino terminal dipeptides ending in proline has been described for canine gastrin releasing peptide (24) and substance P (16). A possible enzyme responsible for this cleavage is a postproline dipeptidyl amino peptidase (EC 3.4.14.1). These data suggest that the shorter form is stored in tissue but we cannot exclude that the smaller form was produced in vitro. However, the postproline dipeptidyl amino peptidase is inhibited at pH 1.5 as are other amino peptidases. This inhibition combined with rapid processing of tissue extracts should prevent in vitro degradation. Our laboratory (12) and the laboratory of Tatemoto (31,32) concurrently purified and characterized the structure of human PYY. The structures presented (12,31) by both groups are identical. Tatemoto et al. used a chemical method for detection of tyrosine-amide (the carboxyl terminus of human PYY) and only one PYY form was purified (32). We have purified and characterized two PYY forms. Our methods of sequence analysis (microsequencing and mass spectral analysis of intact and tryptic fragments) provides repeated confirmation of the structural differences between porcine and human PYY. More importantly, our results show the structure of a new molecular form of human PYY, PYY(3-36). PYY has been proposed to have biological activity for intestinal vasoconstriction, inhibition of gastric acid secretion and pancreatic bicarbonate and protein secretion, and inhibition of gastric emptying and gastrointestinal motility (1, 3, 9, 19, 21). It has been speculated that the enterogastrone-like effect of PYY is vagally mediated (23). Little is known about the structure-function relationships of PYY. It will be important to evaluate if PYY(336) has the same half-life as the longer form. and whether the biological activity of both forms is the same. It will also be interesting to evaluate if the changes at positions 3 and 18 alter the biological activity of PYY. PYY belongs to the PP family of peptides. In human, porcine and rat PYY, NPY and PP, 15 amino acid positions are identical (Fig. 5). Human PYY differs from porcine PYY in two positions. Human PYY varies from human NPY at 12 positions and from human PP in 19. It is interesting that the two differences between

PYY Human

YP I KF_EAPGED~S

Porcine

- -A ................

S ...................

- -A

S . . . . . . . . . . . . . . . . . .

Rat

PEE

LNRY

. . . . . . . . . . . . . .

YAS

L RH~[LN

LVTRQRY

g

NPY Human

--S--DN

.....

PA-DMA---SA

....

I--I

Porcine

--S--DN

.....

PA-D-A---SA

....

I---I .....

Rat

--S--DN

.....

PA-DMA---SA

....

I--I

......

.....

PP Human

A- LE-VY-

-DN-

T- - QMAQ-A-D-

-R-

I-ML-

-P-

Porcine

A-LE-VY-

-DD-

T--QMAQ-A-E-

-R-

I-ML-

-P- -

Rat

A- LE-MY-

- DY-

TH-

QRAQ-

ETQ-

-R..I -TL-

-P-

-

FIG. 5. Structures of human PYY(1-36) compared to porcine (28) and rat (10) PYY(1-36), human (20), porcine (30) and rat (4,10) NPY(I-36), and human (6,7), porcine (6) and rat (17) PP. Only human PYY has also been shown to exist in a smaller form [human PYY(3-36)]. Differences in sequence from human PYY are shown for the other peptides. Positions identical in PYY, NPY and PP are underlined.

human and porcine PYY occur in positions that also vary between human PYY, NPY and PP. It is possible that these positions do not affect binding to receptors, but this evaluation awaits use of human PYY in radioreceptor assays. The most highly conserved region among all these peptides is at the carboxyl terminus where 4 of 5 residues are identical (Fig. 5). lnui (15) and Balasubramaniam (5) have both shown that carboxyl terminal fragments of PYY will bind to receptors. Carboxyl terminal fragments 17-36 and 24-36 of PYY bind to a lesser degree [25 and 285 times less well than PYY(1-36), respectively] to brain membranes (15). Walker and Miller (34) found PYY(13-36) displaced radiolabeled PYY about 10 times less potently from rat brain membranes than the intact peptide. Carboxyl terminal fragment (22-36) of PYY is 25 times less potent for displacement of '25I-pYY from rat intestinal epithelial plasma membranes, whereas the amino terminal fragment 1-28 bound 15,000 times less potently than intact PYY (5). It is not known if deletion of two amino acids from the amino terminus of PYY affects receptor binding or biological activity. However, deletion of the amino terminal tyrosine from the structurallyrelated peptide NPY resulted in marked reduction in vasoconstrictor potency (25). It is known that PYY is a good competitive ligand for the NPY receptor (34). PYY and NPY have a high degree of homology at the amino terminus. Knowledge of the new molecular form of human PYY, PYY(3-36) will allow its synthesis for immunological and biologicai studies. Differences may be observed between human PYY(336) and PYY(1-36) in the ability to bind to central or peripheral receptors.

ACKNOWLEDGEMENTS

We thank Mrs. I. Neumann for excellent technical assistance. This research was supported by the grant EY 14/2-3 of the Deutsche Forschungsgemeinschaft, by NIH grants DK 17294, 33850 (J.R.R.) and DK 33155 (J.E.S.) and the Veterans Administration.

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