Evidence for a novel FMRFamide-related heptapeptide in the pulmonate snail Siphonaria pectinata

Pepttde*, Wol 8, pp 533-538 ~ Pergamon Journals L t d , 1987 Printed m the U S A

01%-9781/87 $3 00 + 00

Evidence for a Novel FMRFamide-Related Heptapeptide in the Pulmonate Snail

Siphonaria pectinata D. A. P R I C E , 1 C H A R L E S G. C O B B , K. E. D O B L E , J O H N K. K L I N E A N D M. J. G R E E N B E R G C.

W.

Whitney Laboratory, University o f Florida, Rt. 1, Box 121, St. Augustine, F L 32086 R e c e i v e d 13 J a n u a r y 1987

PRICE, D A , C. G COBB, K E DOBLE, J K KLINE AND M J. GREENBERG Evlden~efi~ra novel FMRFam~derelated heptapeptMe m the pulmonate snad Slphonana pectlnata PEPTIDES 8(3)533-538, 1987 --Extracts of whole false limpets (Stphonarta pe~ tmata) were analysed to determine their complement of FMRFamlde-related peptldes. As m other pulmonates, FMRFamlde itself was found to account for only a portion of the immunoreactlvlty, the largest lmmunoreactlve peptide peak eluted during HPLC under acidic conditions at the same posltlon as a peak also found m other pulmonates. This major peak was resolved into two components by HPLC at neutral pH, and one component was identified as the heptapeptide amlde, GDPFLRFamlde, previously described from Lymnaea The amino acid composition of the second component indicates that it is also a heptapeptlde, but that it has two Asx (aspamc acid or asparagmyl) residues instead of the one found m the previously ~dentlfied pulmonate heptapeptldes FMRFamlde

FMRFamlde-related heptapeptldes

Pulmonate molluscs

S~phonarta

G D P F L R F a m I d e from Helir [12], and the lack of p Q D P F L R F a m i d e in Lymnaea suggests that the distribution of FMRFam~de-related peptides in the suborders of Pulmonata are characteristic of these groups. To explore this hypothesis further, we began to investigate this peptide family m the primitive basommatophoran Siphonarta peettnata. These "false limpets" are members of the superfamily Siphonariacea which, with the Amphibolacea, diverged very early from the main basommatophoran stem [3]. Consistent with its phylogeny, we report that Stphonarta contains, in addition to FMRFamide, one FMRFamide-like heptapeptide (GDPFLRFamide) in common with the higher hmnic basommatophoran Lymnaea. However, another heptapeptide, hitherto unknown, is also present.

T H E sequence of FMRFamlde (Phe-Met-Arg-Phe-NH2) was determined and confirmed 10 years ago in a bivalve mollusc [10] Since that time this neuropeptide has been identified m the ganglia of many species representative of the major molluscan taxa. We have therefore concluded that FMRFamide is ubiquitous m the phylum Mollusca (reviewed in [8]). The pulmonate gastropods are exceptional among molluscs in that they contain, not only F M R F a m id e itself, but also substantial amounts of two additional FMRFamiderelated peptides. Information about these additional peptldes has been obtained from species representative of the two major suborders of pulmonates, Stylommatophora and Basommatophora. First, in Hehx aspersa and related stylommatophoran terrestrial snails, fractionatlon of ganglion extracts by HPLC reveals three large peaks detectable by radioimmunoassay (RIA). Two of these peaks are known: FMRFamide and the heptapeptide pGlu-Asp-Pro-Phe-LeuArg-Phe-NH2 (pQDPFLRFamide) [11]; the third is not yet characterized, but also appears to be a heptapeptlde [12]. In contrast to those of Helix, the sequence of the two additional peptides in the basommatophoran aquatic snail Lymnaea stagnahs have been shown to be the Ser 1 and Gly 1 analogs of p Q D P F L R F a m i d e : respectively, S D P F L R F a m i d e and G D P F L R F a m I d e [1,2]. The absence of both SDPFLRFamide and

METHOD

Animals, Dissection and Extraction False limpets (Stphonarta pectmata) were collected from coquina rocks in the intertidal zone just south of Marineland on the east coast of Florida. The animals were very abundant but they are also very small, so no attempt was made to dissect out their ganglia. Instead, the snails--shells and all---were put directly into acetone (about four volumes of acetone per volume of snails) and the mixture was put in the

1Requests for reprints should be addressed to Dr D A. Price, The WhRney Lab, Rt. 1, Box 121, St Augustine, FL 32086

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P R I C E ET AL.

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Frochon No FIG 1 Fractlonatlon of a snad extract on Sephadex G-15 The column (2 1x45 cm) was equlhbrated and eluted with 0 ! M acetic acid The flow rate was approximately 1 ml/mln, and 10 minute fractions were collected An aliquot (2 #1) of each fraction was taken for RIA and used to compute the nmol equivalents of FMRFamlde/fractlon V ~= 156 ml

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FIG. 2 Further fractlonatlon of the fir,t Sephadex peak (fl actions 12-18 m Fig I ) on HPLC The column (Waters C18 Radlal-Pak) was eluted for the first 10 minutes with a buffer (0 5 M ammomum acetate m 0.1 M acetic acid) contalmng 4%. n-butanol, and then with a hnear gradient of n-butanol up to 8% at 30 rain The flow rate was 4 ml/mln and 0 5 minute fractions were collected. An aliquot (2/zl) of each fraction was taken for RIA and used to compute the nmol equivalents of FMRFamide/fractlon The elutlon t~mes of some relevant synthetic peptldes, determined from separate but similar runs, are shown by arrows.

freezer. T h e a n i m a l s were a l l o w e d to s t e e p in the a c e t o n e for v a r y i n g t i m e s ( o v e r n i g h t to m a n y d a y s ) w i t h o c c a s i o n a l s h a k ing to mix the c o n t e n t s . S e v e r a l b a t c h e s o f snails (1000-3000 i n d i v i d u a l s e a c h ) w e r e e x t r a c t e d in this way. A f t e r e x t r a c t i o n , t h e cold a c e t o n e w a s d e c a n t e d a n d centrifuged. T h e s u p e r n a t a n t s w e r e p o o l e d a n d t h e a c e t o n e rem o v e d o n a r o t a r y e v a p o r a t o r . T h e small v o l u m e o f a q u e o u s s o l u t i o n r e m a i n i n g in t h e r o t a r y e v a p o r a t o r flask was f o r c e d t h r o u g h a small C18 c a r t r i d g e ( W a t e r s S e p - P a k ) w h i c h w a s t h e n w a s h e d with w a t e r ; finally, t h e a c t i v e m a t e r i a l w a s e l u t e d with m e t h a n o l . T h e m e t h a n o l w a s r e m o v e d ( r o t a r y e v a p o r a t o r ) , a n d t h e r e s i d u e was e i t h e r t a k e n u p m 0.1 M

a c e t i c acid for a p p l i c a t i o n to a S e p h a d e x G-15 column, or t a k e n up in a m m o n i u m a c e t a t e b u f f e r for H P L C in t h e b u t a n o l b u f f e r s y s t e m d e s c r i b e d below.

Gel Chromatography (Sephadex G-15) T h e c o l u m n (2 1 x 4 5 cm) w a s e q u i l i b r a t e d a n d eluted with 0.1 M acetic acid at a flow rate o f a p p r o x i m a t e l y 1 ml/min. T h e s a m p l e v o l u m e was 3-5 ml, a n d 10 m i n u t e f r a c t i o n s were collected, A n a l i q u o t ( 2 / z l ) o f e a c h f r a c t i o n was t a k e n for R I A , a n d the a c t i v e f r a c t i o n s c o n s t i t u t i n g the large p e a k of i m m u n o r e a c t l v l t y eluting at a p p r o x i m a t e l y o n e c o l u m n vol-

A NOVEL FMRFAMIDE-RELATED HEPTAPEPTIDE

535 combined and lyophdlzed for further H P L C in either the trifluoroacetic acld/acetomtrile (TFA/ACN) system or the phosphate/acetonitrde system.

H P L C TFA/ACN Sy,stem

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The column (Waters C18 p~-Bondapak) was eluted with an aqueous solvent of water containing 0 072% tnfluoroacetlc acid, and an orgamc solvent of 8(F~ acetomtrlle in water containing about the same concentration of acid (In practice th~s was achieved by adding acid to the organic solvent until its UV absorbance (210 nm) matched that of the aqueous) The solvent was pumped at 2 ml/mln, and the gradient was from 12% acetomtrlle at the start, to 4(F/~ at 24 minutes. Fractions (0 5 minute) were collected and ahquots (2 /xl) taken for radxoimmunoas,say. Actwe fractions were either combined for further HPLC, or used for amino acid analysis as described below.

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FIG. 3 The major peak from a fractlonatlon similar to that shown m Fig 2, but without a prehmlnary Sephadex fractlonatlon, was applied to a /x-Bondapak C18 column (Waters) and eluted with a tnfluoroacet~c ac~d/acetomtnle gradient system (see the Method section) The solvent was pumped at 2ml/mm, and the gradient was, from 12% acetonltnle at the start, to 40% at 24 mm. Fractions (1 ml) were collected, and ahquots (2/~l) were taken for radloRmmunoassay, the remainder of each fraction was hydrolyzed for amino acid analys~s Only the region of the chromatogram containing the major peak IS shown. The top trace is the UV absorbance at 210 nm, the ~mmunoreactlvlty and amino acid levels are shown below

ume were combined and run through a Sep-Pak with a large syringe. The active material was eluted from the Sep-Pak with methanol, the methanol was removed on a rotary evaporator, and the residue, in buffer, was applied to H P L C in the butanol buffer system.

H P L C Butanol System The column (Waters C18 Radial-Pak) was eluted at 4 ml/min with 0.5 M ammonium acetate (pH 5.5)/n-butanol buffers The initial buffer (40 ml/l n-butanol) was run for 10 minutes, after which a 20 minute gradient to the final buffer (80 ml/1 n-butanol) was started. Fractions (0.5 minute) were collected, and an aliquot (2 /zl) of each was taken for radioimmunoassay. The fractions making up each peak were

A Nova-Pak C18 (Waters) column was used. The flow rate was 2 ml/mm, and UV absorbance was monitored at 210 nm. The aqueous solvent was 5 mM phosphate buffer, pH 7.0 (made from the monobaslc sodmm salt adjusted with NaOH), and a linear gradient was run, from 18% acetonltrile at the start, to 48% at 30 mln. Half minute (1 ml) fractions were again collected. After aliquots had been taken for RIA, the remainder of each fraction was hydrolyzed (18 hours at 108°C in 6 N HCI under nitrogen) for amino acid analysis.

The Radmunmunoa~sav (RIA ) The antiserum was raised in rabbits to a conjugate of Tyr-Gly-Gly-FMRFamlde (YGGFMRFamlde) and thyroglobuhn; I J2~-YGGFMRFamide was the trace. The antiserum has been described [7]. The assay was performed as follows An aliquot (2 /xl) of each fraction was transferred to a glass test tube with an automatic diluter (Micromedic model 30010) that added RIA buffer (48/zl) to give a sample volume of 50/xl. The RIA buffer contains 0.01 M phosphate (dibasic sodmm salt), 0.9% sodium chloride, 1% bovme serum albumin, 0.01% merthiolate, and 0.025 M sodium EDTA and Is adjusted to pH 7.5 with sodium hydroxide An otherwise identical buffer, but wtthout albumin, was used for sample dilution to prevent small amounts of BSA on the tip of the diluter from contaminating the HPLC fractions. The trace (in 100/zl of buffer) was added together with diluted antiserum (also 100/~1) to each tube; the final dilution of antiserum was about 1.60,000, and there were 10,000 CPM of trace (Price [8]) All of the tubes were refrigerated overmght (4°C), and charcoal solution (1 ml) was added in the morning. (Charcoal solution contains 0.25% charcoal, 0.025% dextran, and 0.01% merthiolate m 0 1 M sodmm phosphate buffer (87 ml/100 ml 0.1 M Na2HPO~ and 13 ml/100 ml 0.1 M NaH2PO4 pH 7 5, it was stirred overnight before its first use and was kept refrigerated thereafter.) The charcoal was centrifuged down (2500 × g for 15 minutes) after 15 mmutes of mcubation; the supernatants were then decanted and counted (LKB-Wallac MmiGamma 1275) RESULTS Two major peaks of lmmunoreactivity were separated by the Sephadex column (Fig. 1). The first peak elutes at 2.5-3 void volumes, the elution position of all the known FMRFamide-hke peptides. The second peak is much more

P R I C E ET AL.

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'°'° I FIG 4 The major peak (47-51) from Fig 2 (HPLC, butanol system) was rerun on a Nova-Pak C 18 column (Waters) with a phosphate/ACN buff'el system (see the Method sectmn) The left uace shows the UV absorbance at 210 nm for a mixture of three synthetm pepUdes 2 nmol each of GDPFLRFamlde and SDPFLRFamlde, ,rod 5 nmol of pQDPFLRFamlde The right trace shows the UV absorbance of the peak, the material elutlng at 14 54 minutes wa~ not lmmunoreacttve

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FIG. 5 Characterization of fractions from the HPLC run shown in Fig 4. (A) The immunoreactlvlty was determined In an ahquot (2/~l of each fraction). (B) The remainder of each fraction was hydrolyzed and subjected to amino amd analysis The profiles for selected amino amds are shown

retained (5 to 6 void volumes) and contains very httle peptide. A v e r y similar peak is seen in Hehx, and L e h m a n and Price [6] have argued that it is a non-peptlde R I A artifact. W h e n the first S e p h a d e x peaks from different batches w e r e put through the butanol H P L C system, similar arrays o f F M R F a m i d e - l i k e i m m u n o r e a c t l v i t y appeared (Fig. 2). T w o major peaks were consistently resolved, one co-eluted with F M R F a m i d e or (as illustrated in Fig 2) its oxidation product, and the other eluted at the position of S D P F L R F a m I d e and G D P F L R F a m i d e which co-elute in this H P L C system. No peak eluted at the e x p e c t e d p o s m o n of p Q D P F L R F a m i d e (31 minutes; not shown m Fig 2) When the a c e t o n e extract was directly applied to the H P L C without prior S e p h a d e x c h r o m a t o g r a p h y , a large additional peak

of i m m u n o r e a c t i v l t y was seen j u s t before the position of the S D P F L R F a m l d e / G D P F L R F a m l d e pair (data not shown). As e x p e c t e d , w h e n the F M R F a m i d e peak was rerun in the T F A / A C N system, it again gave rise to a peak at the position of F M R F a m l d e with an additional peak at the position of oxidized F M R F a m i d e . Further, when the oxidized F M R F a m l d e peak was rerun, it eluted again at the position of oxidized F M R F a m i d e (not shown). The peak corresponding to the S D P F L R F a m l d e / G D P F L R F a m i d e pair also eluted again with S D P F L R F a m i d e and G D P F L R F a m i d e in the T F A / A C N system. H o w e v e r , the U V peak at this elutlon position is clearly not symmetrical; a shoulder on the leading edge (Fig. 3) indicates that it contains at least two substances. W h e n the fractions across this peak were analyzed

A NOVEL FMRFAMIDE-RELATED HEPTAPEPTIDE TABLE 1

DISCUSSION

A M I N O ACID A N A L Y S I S OF I M M U N O R E A C T I V E P E A K F R A C T I O N S F R O M SIPHONARIA

Fraction 17

537

Fraction 24

Amino Aod

nmol

Ratio

nmol

Ratio

Glycme Aspartlc Acid Prohne Phenylalanlne Leucme Argmme Serlne

0 361 1.321 0.697 1 298 0 802 0 705 0 203

0 52 1 90 1 00 1 86 1.15 1 01 0 29

0 476 0 525 0 439 0 787 0.510 0 482 0 119

1 08 1 20 1 00 1 79 1.16 1 10 0 27

Fractions 17 and 24 from the first and second peaks, respectively, in Fig 5 (HPLC. phosphate/ACN system) were hydrolyzed in redlstdled, constant bolhng HCI at 108°C for 16 hours in a sealed glass tube under nitrogen The amino acids were determined with an automatic analyzer (Hitachi 835)

for ammo acid content, we saw that the levels of Arg, Phe, Leu, Gly and Pro followed the immunoreactive content, but the level of serlne did not (Fig. 3) indicating that the peptide SDPFLRFamlde is probably not present. The aspartic acid profile of this peak is especially notable it is coincident with the peak of immunoreactwity, but its shape is different, and it is much bigger than would be expected if it were due either to SDPFLRFamIde or GDPFLRFamlde. An attractive interpretation Is that the leading shoulder in the UV peak is an analog of GDPFLRFamide with two Asx (aspartyl or asparingyl) residues Once we had an indication that the major peak in the TFA/ACN system contained two pept~des, we used the ACN/phosphate system in an attempt to resolve them since earlier work on Lymnaea [2] had shown that this system separates SDPFLRFamide from GDPFLRFamlde. At first we continued to use the TFA/ACN step also, but our recoveries were poor Therefore, we omitted the TFA/ACN step and went directly from the butanol system to the ACN/phosphate and found two peaks of immunoreactlvlty (Fig. 4). One of these peaks elutes at the position of standard GDPFLRFamlde, while the other elutes between standard pQDPFLRFamide and SDPFLRFamide. The amino acid composition was determined for each fraction from the start of the first peak to the end of the second. The profiles of 5 relevant amino acids are depicted in Fig 5, and the complete compositions of the peak fractions are hsted m Table 1 The amino a o d profiles of the two immunoreactive peaks are clearly different. First, for neither peak does the level of serine follow the profile of lmmunoreactivity, confirming our finding with the TFA/ACN system which showed that SDPFLRFamlde is not present in these extracts. Second, the immunoreactive peak eluting w~th standard GDPFLRFam~de has a composition consistent with its being GDPFLRFamlde (fraction 24 in Table 1; F~g. 5). Finally, the unidentified first peak of immunoreactxvity contains levels of phenylalanlne and aspartlC acid that are equal and about twice as large as the levels of the other amino acids expected (Le , Pro, Leu, and Arg, Fig. 5 and fraction 17 in Table 1). The composition of this peak seems to be: Asx2Phe.,ProlLeulArg~.

We have identified, in this report, the three major FMRFamide-related peptides in extracts of the primitive basommatophoran pulmonate snail Stphonarla pectinata. First, FMRFamide itself is present, supporting our conclusion that this tetrapeptide occurs m all molluscs [4, 8, 9]. Second, the extracts contain GDPFLRFamide, but not pQDPFLRFamide, confirming our hypothesis that the distribution of the FMRFamide-related peptides in the pulmonates reflects the phylogeny of these molluscs In particular, pQDPFLRFamlde appears to be a specialization of the Stylommatophora, whereas GDPFLRFamide is characteristic of the Basommatophora. But this pattern is not sufficiently established, more species should be examined. Finally, we have discovered a hitherto unidentified heptapeptide in Stphonarul with the composition' Asx..,Phe,,Pro: LeulArgl. The sequence of this peptide is now being investigated, but by analogy with pQDPFLRFamide, we expect that it will be Asn-Asp-Pro-Phe-Leu-Arg-Phe-NH2. The FMRFamide-related heptapeptldes of pulmonates have the common sequence. X-Asp-Pro-Phe-Leu-Arg-PheNH.,. Thus their diversity seems to reside solely with the N-terminal residue. Nevertheless, that diversity is already quite extensive, especially considering the paucity of studied species [ 1, 2, 8]. The finding of yet another member of the family suggests that the end is not yet in s~ght Similar heptapeptldes have not been found in any non-pulmonate mollusc, though the enkephahn-related heptapeptlde YGGFMRFamlde has been reported from O~topu,s [14]. Conversely, FLRFamlde, an analog of the pulmonate heptapeptldes, has been detected at low levels m most molluscs examined [8, 9, 15], but not yet m any pulmonate. The array of FMRFamlde-related peptides in Stphonaria is similar m ~ts composition to those found in every puimonate studied, i.e., three major c o m p o n e n t s - - F M R F a m i d e and a pair of heptapeptides--all at roughly the same level. That these arrays have group functions in the organism, integrating the various transmittal or hormonal roles of its three component peptides, seems likely. The matter has been discussed by Lehman and Greenberg [5] in terms of myocardial and somatic muscular responses that influence the retraction and emergence of Hehx from its shell. Price [8] has speculated about the evolution of the FMRFamiderelated peptldes of pulmonates, but the genetic basis of these peptldes was, unfortunately, not elucidated by the description of the gene that encodes the FMRFamlde precursor of Aplyma ~ahfornt~ a [13]. Two observations reported here are of technical importance. First, we were able to recover significant quantities of FMRFamlde-like immunoreactlvity--several picomoles per animal--simply be steeping whole snails m acetone. This finding suggests that it will be feasible to examine the peptide content of any species, no matter how small, an essential requirement in phylogenetlc studies The extraction of whole animals has additional advantages: it precludes dissection which, with small animals, requires much more time and many more specimens; and it exploits the high concentrations of peptides often found in organs outside the CNS (e.g., [6]). The second, more important, finding was that we were able to purify the peptides sufficiently in four chromatographic steps to obtain good amino acid analyses. In partlcular, although chromatography on Sephadex G-15 would seem to have been outdated by modem HPLC methods, it

538

PRICE ET AL.

still p r o v i d e s a v a l u a b l e step m the purification o f F M R F a m i d e - l i k e p e p t i d e s . Its p r i m a r y a d v a n t a g e is t h a t all of t h e s e q u e n c e d F M R F a m t d e - r e l a t e d pept~des elute tog e t h e r b e t w e e n 2.5 a n d 3 0 v m d v o l u m e s . N e v e r t h e l e s s , this gel filtration step also r e v e a l e d a s e c o n d m a j o r p e a k o f ~mm u n o r e a c t t v l t y s~milar to o n e o b s e r v e d with H e h x e x t r a c t s b y L e h m a n a n d Price [6]. T h e p e a k r u n s as a single c o m p o n e n t eluting n e a r S D P F L R F a m i d e a n d G D P F L R F a m l d e m e~ther the b u t a n o l or T F A / A C N s y s t e m s . B u t b o t h t h e peptide level o f the p e a k (as e v i d e n c e d b y a m i n o acid a n a l y s i s o r U V a b s o r b a n c e ) a n d its b~ological a c t w i t y [6] are v e r y low m c o m p a r i s o n to its i m m u n o r e a c t i v i t y . A l t h o u g h this p e a k c o u l d be a r t i f a c t u a l [6] - - a c o n c l u s i o n b y no m e a n s c e r t a m - - t h e p h e n o m e n o n is surely w o r t h y o f f u r t h e r i n v e s t i g a t i o n .

ACKNOWLEDGEMENTS Th~s project was supported by NIH grant HL28440 The work of C G Cobb and J K Khen, m particular, was supported by the Grass Foundation whom all of the authors thank for sustaining undergraduate research at th~s institute The assistance of Louise MacDonald, Lynn Mdstead and Jim Netherton m prepanng the manuscript ~s gratefully acknowledged Th~s ~s contHbuhon No 257 from the Tallahassee, Sopchoppy and Gulf Coast Marine B~olog~cal Assocmt~on.

ADDENDUM W e h a v e o b t a i n e d t w o a d d i t i o n a l results t h a t s u p p o r t t h e c o n c l u s i o n s p r e s e n t e d here. First, Dr. B e n M. D u n n ( U m v e r s i t y o f F l o r i d a Protein C h e m i s t r y Core Fac~hty) s y n t h e sized t w o of the s e q u e n c e s p o s s i b l e for the u n k n o w n hept a p e p t i d e b a s e d o n its c o m p o s i t i o n . One o f t h e m , N D P F L R F a m i d e , c o - e l u t e d w~th t h e u n k n o w n pept~de, w h d e the o t h e r , D D P F L R F a m i d e , did not. S e c o n d , we e x t r a c t e d a n o t h e r b a t c h of Stphonarta, purified the t w o heptapet~de p e a k s , a n d sent t h e m to Dr. T e r r y D L e e (Dwis~on o f I m m u n o l o g y , B e c k m a n R e s e a r c h I n s t i t u t e o f t h e C~ty o f H o p e , D u a r t e , CA) for s e q u e n c i n g by F A B m a s s s p e c t r o m e t r y . H e o b t a i n e d m o l e c u l a r ~ons for b o t h h e p t a p e p t i d e s F o r one, the o b s e r v e d m o l e c u l a r weight o f the M H + ion (th~s c o r r e s p o n d s to the m o l e c u l a r weight plus 1) was 850.37 while the c a l c u l a t e d value for G D P F L R F a m l d e is 850.44; th~s s p e c t r u m c o n t a i n e d sufficient s e q u e n c e ions that the e x a c t s e q u e n c e of this p e a k as G D P F L R F a m l d e c o u l d b e c o n f i r m e d . F o r the o t h e r p e a k , the o b s e r v e d v a l u e o f the M H + ion (907.43) a g r e e d well with the c a l c u l a t e d wdue for N D P F L R F a m ~ d e (907.46), and ruled out the poss~bd~tles D D P F L R F a m l d e a n d N N P F L R F a m ~ d e w h i c h are, r e s p e c t w e l y , one d a l t o n h e a w e r or h g h t e r Since a strong s e q u e n c e ~on c o r r e s p o n d i n g to the f r a g m e n t P F L R F a m ~ d e was o b s e r v e d m the F A B mass s p e c t r u m and the N - t e r m i n a l residue was d e t e r m i n e d as a~pa~ aglne IN) by conventional sequencing, the proposed structure N D P F L R F a m ~ d e ~s c o n f i r m e d

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