Insect Myotropic Peptides - ACS Symposium Series (ACS Publications)

Chapter 5 Insect Myotropic Peptides Isolation, Structural Characterization, and Biological Activities 1

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G. Mark Holman , Ronald J. Nachman , Mark S. Wright , Liliane Schoofs , Timothy K. Hayes , and Arnold DeLoof 3

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Downloaded by RUTGERS UNIV on December 30, 2017 | http://pubs.acs.org Publication Date: January 25, 1991 | doi: 10.1021/bk-1991-0453.ch005

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Veterinary Toxicology and Entomology Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, College Station, TX 77840 Department of Entomology, Texas A&M University, College Station, TX 77843 Zoological Institute, Katholiecke University, B-3000, Leuven, Belgium 2

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In the early 1980's, dramatic improvements in both peptide isolation technology and the instrumentation for structural characterization resulted in an avalanche of new insect neuropeptide structures. Of the 50-60 known structures, about half exhibit effects on the contractile activity of insect visceral muscle at physiological concentrations. This report describes the strategies and tactics that were utilized to successfully isolate, purify, and structurally characterize this group of insect neuropeptides. The s i n g l e most i m p o r t a n t r e q u i r e m e n t f o r a s u c c e s s f u l i s o l a t i o n o f a b i o l o g i c a l l y a c t i v e n a t u r a l p r o d u c t i s a r e l i a b l e b i o a s s a y system f o r f o l l o w i n g t h e compounds o f i n t e r e s t t h r o u g h t h e p u r i f i c a t i o n scheme. The s e m i - i s o l a t e d h e a r t o f P e r i p l a n e t a a m e r i c a n a and t h e i s o l a t e d h i n d g u t o f P. a m e r i c a n a o r Leucophaea maderae f u l f i l l t h a t r e q u i r e m e n t as shown by t h e number o f s u c c e s s f u l i n s e c t p e p t i d e i s o l a t i o n s which u s e d one o f those t h r e e p r e p a r a t i o n s as a bioassay. A p p r o x i m a t e l y h a l f o f t h e 60 o r so i n s e c t n e u r o p e p t i d e s whose s t r u c t u r e s a r e known were i s o l a t e d b a s e d upon an e f f e c t ( e i t h e r s t i m u l a t o r y o r i n h i b i t o r y ) upon c o c k r o a c h v i s c e r a l m u s c l e . Our l a b o r a t o r y has u s e d t h e i s o l a t e d h i n d g u t o f t h e c o c k r o a c h , L. maderae. as a b i o a s s a y p r e p a r a t i o n f o r more t h a n 20 y e a r s ( 1 ) . This p r e p a r a t i o n r e q u i r e s m i n i m a l d i s s e c t i o n s k i l l s and t h e a s s o c i a t e d i n s t r u m e n t s a r e easy t o o p e r a t e . I n a d d i t i o n , the hindgut i s p h y s i c a l l y rugged and i f c a r e i s t a k e n t o a s s u r e c o n s t a n t a e r a t i o n , a s i n g l e h i n d g u t w i l l remain v i a b l e f o r an e n t i r e day. A f t e r d i s s e c t i o n and s u s p e n s i o n o f t h e gut i n t h e chamber, a 30 min t o 1 h r p e r i o d o f e q u i l i b r a t i o n i s a l l o w e d d u r i n g which time t h e s a l i n e i s r e p l a c e d 3-5 t i m e s . At that point, a r e l a t i v e l y constant p a t t e r n o f spontaneous c o n t r a c t i o n s i s e s t a b l i s h e d and t h e a s s a y o f f r a c t i o n s f o r a c t i v i t y can begin. A l t e r a t i o n o f the p a t t e r n o f spontaneous c o n t r a c t i l e a c t i v i t y ( e i t h e r s t i m u l a t o r y o r i n h i b i t o r y ) This chapter not subject to U.S. copyright Published 1991 American Chemical Society

Menn et al.; Insect Neuropeptides ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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H O L M A N ET AL.

Insect Myotropic Peptides

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i n d i c a t e s the p r e s e n c e o f a b i o l o g i c a l l y a c t i v e component i n t h a t fraction. When the t e s t s a l i n e i s r e p l a c e d w i t h f r e s h s a l i n e , the e f f e c t s o f b o t h s t i m u l a t o r s and i n h i b i t o r s cease a l m o s t immediately and w i t h i n 2-5 min the spontaneous c o n t r a c t i l e a c t i v i t y r e t u r n s t o a p r e - e x p o s u r e p a t t e r n and the gut i s ready t o a c c e p t a n o t h e r fraction. D u r i n g our work on the i s o l a t i o n o f the c o c k r o a c h , c r i c k e t , and l o c u s t p e p t i d e s , up t o 80 samples/day were e v a l u a t e d on a s i n g l e h i n d g u t p r e p a r a t i o n . W i t h few e x c e p t i o n s ( n o t e d l a t e r ) , the r e c e p t o r s o f the L. maderae h i n d g u t as w e l l as the h e a r t and h i n d g u t o f P. americana were q u i t e s e n s i t i v e t o many o f the i n s e c t m y o t r o p i n s and c o n s e q u e n t l y o n l y s m a l l q u a n t i t i e s o f m a t e r i a l were expended d u r i n g p u r i f i c a t i o n p r o c e d u r e s . The remainder o f t h i s r e p o r t d e s c r i b e s how t h e s e v i s c e r a l muscle b i o a s s a y s were used t o s u c c e s s f u l l y i s o l a t e and s t r u c t u r a l l y c h a r a c t e r i z e more t h a n two dozen i n s e c t n e u r o p e p t i d e s . C a r d i o a c c e l e r a t o r y Neuropeptides

o f P.

americana

I n l a t e 1984, two independent r e s e a r c h teams a l m o s t s i m u l t a n e o u s l y p u b l i s h e d the s t r u c t u r a l c h a r a c t e r i z a t i o n and s y n t h e s i s o f two m y o t r o p i c n e u r o p e p t i d e s i s o l a t e d from P. a m e r i c a n a c o r p o r a c a r d i a c a ( c c ) e x t r a c t s (.2,3). Both teams used r e v e r s e - p h a s e , h i g h p e r f o r m a n c e l i q u i d chromatography (RP-HPLC) w i t h i o n - p a i r i n g r e a g e n t s t o o b t a i n pure p e p t i d e s f o r a n a l y s i s . A C-18 reverse-phase column was u s e d by b o t h teams and p u r i f i c a t i o n was a c c o m p l i s h e d w i t h a s o l v e n t programmed r u n f o l l o w e d by an i s o c r a t i c r u n o f the a c t i v e peaks. I n one c a s e , the two p e p t i d e s were i s o l a t e d b a s e d on t h e i r s t i m u l a t o r y a c t i o n on a l o c u s t somatic muscle p r e p a r a t i o n ( 2 ) . In the second c a s e , s t i m u l a t i o n o f the s e m i - i s o l a t e d P. a m e r i c a n a h e a r t was u s e d t o d e t e c t a c t i v e HPLC peaks and f r a c t i o n s ( 3 ) . Mass s p e c t r a l a n a l y s i s o f the i n t a c t p e p t i d e s (2) o r t h e i r c h y m o t r y p t i c fragments (3) was u s e d t o deduce the sequences. Sequence methods u t i l i z i n g Edman c h e m i s t r y were p r e c l u d e d s i n c e b o t h p e p t i d e s e x h i b i t e d a b l o c k e d amino terminus (enzymatic d e b l o c k i n g p r o c e d u r e s had n o t been d e v e l o p e d a t t h i s t i m e ) . The s t r u c t u r e s o b t a i n e d by b o t h groups were: M-I ( 2 ) , CC-I (3) (Pea-HGH-I)

pQVNFSPNWamide

M-II ( 2 ) , CC-II (3) (Pea-HGH-II)

pQLTFTPNWamide

B o t h o f t h e s e p e p t i d e s e x h i b i t e d c o n s i d e r a b l e sequence i d e n t i t y w i t h the l o c u s t a d i p o k i n e t i c hormone (Lom-AKH-I) and the c r u s t a c e a n r e d p i g m e n t - c o n c e n t r a t i n g hormone (RPCH), as had been s u g g e s t e d by c h r o m a t o g r a p h i c b e h a v i o r , N - t e r m i n a l a n a l y s i s , and amino a c i d a n a l y s i s (3,4). Indeed, b o t h p e p t i d e s were shown t o be f a r more p o t e n t as s t i m u l a t o r s o f h y p e r g l y c a e m i a t h a n as m y o t r o p i n s ( 3 ) . Based upon s i m i l a r i t y o f s t r u c t u r e and a c t i v i t y , Pea-HGH-I and I I a r e c l a s s i f i e d as members o f the AKH/RPCH f a m i l y o f n e u r o p e p t i d e s . A s i n g l e p a i r o f c c c o n t a i n e d about 100 pmol and 40 pmol o f Pea-HGH-I and I I , r e s p e c t i v e l y . The r e l a t i v e l y h i g h t i t e r s o f t h e s e two p e p t i d e s was i n d e e d f o r t u i t o u s as n e i t h e r o f the m y o t r o p i c



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INSECT NEUROPEPTIDES: CHEMISTRY, BIOLOGY, AND

ACTION

b i o a s s a y s was p a r t i c u l a r l y s e n s i t i v e t o t h e s e compounds. The t h r e s h o l d o f a c t i v i t y c o n c e n t r a t i o n f o r the l o c u s t h i n d l e g p r e p a r a t i o n was r e p o r t e d t o be about 100 nM ( 2 ) , w h i l e the t h r e s h o l d c o n c e n t r a t i o n on the i s o l a t e d c o c k r o a c h h e a r t was d e t e r m i n e d t o be about 10 nM w i t h c o n c e n t r a t i o n s o f 100 nM e v o k i n g a 30% i n c r e a s e i n heartbeat rate (3). I n 1989, a t h i r d c a r d i o a c c e l e r a t o r y p e p t i d e was i s o l a t e d from P. a m e r i c a n a c c e x t r a c t s and s t r u c t u r a l l y c h a r a c t e r i z e d ( 5 ) . C o r a z o n i n , pQTFQYSRGWTNamide, was p u r i f i e d w i t h RP-HPLC on a C-18 column w i t h two s o l v e n t systems which d i f f e r e d o n l y w i t h r e s p e c t t o the i o n - p a i r i n g r e a g e n t . The N - t e r m i n a l p y r o g l u t a m i c a c i d (pQ) was removed e n z y m a t i c a l l y and the p r i m a r y sequence o f the 2-11 fragment was o b t a i n e d by gas-phase s e q u e n c i n g . The p r e s e n c e o f a C - t e r m i n a l amide was e l u c i d a t e d by s u b j e c t i n g the C - t e r m i n a l t e t r a p e p t i d e ( o b t a i n e d from a t r y p s i n d i g e s t o f c o r a z o n i n ) t o f o u r c y c l e s o f manual Edman d e g r a d a t i o n f o l l o w e d by the i d e n t i f i c a t i o n o f PTC-Asn-amide w i t h HPLC. C o r a z o n i n was p r e s e n t a t a much lower t i t e r (2 p m o l / p a i r c c ) t h a n the c a r d i o a c c e l e r a t o r s Pea-HGH-I and I I whose t i t e r s were r e p o r t e d t o be 100 pmol- and 40 p m o l / p a i r c c , r e s p e c t i v e l y ( 3 ) . However, c o r a z o n i n was about 1 0 0 - f o l d more p o t e n t t h a n Pea-HGH-I and II. C o n c e n t r a t i o n s o f c o r a z o n i n as low as 0.2 nM evoked s i g n i f i c a n t increases i n heartbeat rate. S t r u c t u r a l l y , c o r a z o n i n does not seem t o f i t i n t o any o f the known i n s e c t n e u r o p e p t i d e f a m i l i e s . The p r e s e n c e o f a p o l a r a r g i n i n e r e s i d u e and the absence o f a Phe r e s i d u e a t p o s i t i o n 4 e x c l u d e c o r a z o n i n from the AKH f a m i l y , w h i l e the absence o f the C - t e r m i n a l pentamer, Phe-X-Pro-Arg-Leu-NH , e x c l u d e s c o r a z o n i n from the l e u c o p y r o k i n i n p e p t i d e f a m i l y d i s c u s s e d l a t e r i n t h i s report. 2

Proctolin I n 1967, Brown (6) d e s c r i b e d a m y o t r o p i n p r e s e n t i n P. a m e r i c a n a h i n d g u t s t h a t s t i m u l a t e d the c o n t r a c t i o n s o f an i s o l a t e d h i n d g u t o f the same s p e c i e s . T h i s m y o t r o p i c p e p t i d e , which we now know as p r o c t o l i n (RYLPT-OH), was s u b s e q u e n t l y i s o l a t e d (7) and s t r u c t u r a l l y c h a r a c t e r i z e d (8) a f t e r a m u l t i - y e a r e f f o r t . A whole-body e x t r a c t t o t a l i n g 125 kg o f P. americana was p r o c e s s e d w i t h contemporary p u r i f i c a t i o n t e c h n i q u e s (open column ion-exchange, a d s o r p t i o n , and t h i n - l a y e r chromatography) t o o b t a i n 180 fig o f p u r e p e p t i d e w h i c h was s u f f i c i e n t f o r s t r u c t u r a l c h a r a c t e r i z a t i o n w i t h hand s e q u e n c i n g methods. D u r i n g the p a s t 14 y e a r s , p r o c t o l i n has been the f o c u s o f numerous p h y s i o l o g i c a l , p h a r m a c o l o g i c a l , immunocytochemical, and b i o c h e m i c a l s t u d i e s . P r o c t o l i n i s w i d e l y d i s t r i b u t e d among the arthropods but i t s presence i n other p h y l a i s not w e l l e s t a b l i s h e d . A l t h o u g h i n i t i a l l y d e s c r i b e d as a n e u r o t r a n s m i t t e r , r e c e n t e v i d e n c e s u g g e s t s t h a t p r o c t o l i n a c t s as a n e u r o m o d u l a t o r and perhaps a l s o as a neurohormone. A thorough and u p - t o - d a t e r e v i e w on the s t a t u s o f p r o c t o l i n r e s e a r c h was r e c e n t l y p u b l i s h e d ( 9 ) .


5.

H O L M A N ET AL.


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H i n d g u t - s t i m u l a t i n g P e p t i d e s o f L. maderae Because o f the complex n a t u r e o f the sample m a t r i x , a f o u r - c o l u m n HPLC p u r i f i c a t i o n system was d e v e l o p e d t o p u r i f y the m y o t r o p i c p e p t i d e s from whole head e x t r a c t s o f L. maderae ( 1 0 ) . A f t e r i n i t i a l f r a c t i o n a t i o n on a p h e n y l r e v e r s e - p h a s e column, a c t i v e samples were s e q u e n t i a l l y p a s s e d t h r o u g h C - l and C-18 r e v e r s e - p h a s e columns. F i n a l p u r i f i c a t i o n was a c c o m p l i s h e d on an 1-125 p r o t e i n s e p a r a t i o n column o p e r a t e d i n a normal-phase mode. The C - l column p r o v i d e d the breakaway s t e p as more t h a n 95% o f the UV-214 a b s o r b i n g m a t e r i a l i n the p h e n y l column f r a c t i o n s was r e t a i n e d l e s s t h a n 24 min b u t a l l o f the m y o t r o p i n s were e l u t e d a f t e r 26 min p o s t i n j e c t i o n . A d d i t i o n a l l y , the C - l column s e p a r a t e d some o f the m y o t r o p i n s t h a t would have been q u i t e d i f f i c u l t t o r e s o l v e on C-18. F o r example, l e u c o k i n i n I (10) and l e u c o p y r o k i n i n (11) were i s o l a t e d i n the same 2-min f r a c t i o n from the p h e n y l column. On the C-18 column, t h e i r r e t e n t i o n times d i f f e r e d by 36 sec and the two peaks were o n l y p a r t i a l l y r e s o l v e d (Holman, u n p u b l i s h e d o b s e r v a t i o n ) . However, the p r o b l e m o f s e p a r a t i n g l e u c o k i n i n I (LK-I) and l e u c o p y r o k i n i n (LPK) n e v e r m a t e r i a l i z e d as LPK was r e t a i n e d 20 min l o n g e r on C - l t h a n LK-I. S i m i l a r l y , l e u c o k i n i n V I I I ( L K - V I I I ) and l e u c o s u l f a k i n i n (LSK) were c o l l e c t e d i n the same p h e n y l column f r a c t i o n (12,13) and e x h i b i t e d v e r y s i m i l a r r e t e n t i o n times (51.4 min and 51.0 min, r e s p e c t i v e l y ) on C-18. However, the problem o f s e p a r a t i o n n e v e r m a t e r i a l i z e d because LK-VIII and LSK were c o l l e c t e d from C - l i n f r a c t i o n s t h a t were s e p a r a t e d by 15 min. The 1-125 p r o t e i n column ( o p e r a t e d i n a normal-phase mode) was chosen as the f i n a l p u r i f i c a t i o n s t e p f o r two r e a s o n s : 1) the s m a l l (sometimes i n v i s i b l e ) amount o f r e s i d u e r e m a i n i n g a f t e r the p r e v i o u s p u r i f i c a t i o n s t e p s was e a s i l y d i s s o l v e d i n 95% a c e t o n i t r i l e : 5 % water ( t h e i n i t i a l - c o n d i t i o n s s o l v e n t f o r t h i s system); and 2) the peaks c o l l e c t e d from t h i s column were i n a s o l u t i o n (75%-85% a c e t o n i t r i l e ) s u i t a b l e f o r p e p t i d e s t a b i l i z a t i o n and r e q u i r e d no f u r t h e r m a n i p u l a t i o n ( o t h e r t h a n c a p p i n g ) p r i o r t o s t o r a g e . The s t r u c t u r e s o f the 11 m y o t r o p i c c o c k r o a c h p e p t i d e s i s o l a t e d w i t h t h i s method a r e l i s t e d , along with appropriate references, i n Table I (leucokinins I - V I I I ) , T a b l e I I ( l e u c o s u l f a k i n i n s ) , and T a b l e I I I (leucopyrokinin). Three s e p a r a t e 3000-head e x t r a c t s were r e q u i r e d f o r the i s o l a t i o n and c h a r a c t e r i z a t i o n o f the Leucophaea p e p t i d e s . N i n e o f the p e p t i d e s o b t a i n e d from the f i r s t 3000-head e x t r a c t were s u b m i t t e d t o a n a l y s i s by mass s p e c t r o m e t r y b u t no u s e f u l d a t a were o b t a i n e d from t h a t e f f o r t . An a d d i t i o n a l 1500 head e q u i v a l e n t s o f one p e p t i d e ( l a t e r named LSK-II) from the second 3000-head e x t r a c t were s u b m i t t e d t o mass s p e c t r o m e t r y w i t h the same r e s u l t . L e u c o k i n i n s I - V I I I (10,12,14,15) and the two LSK's (13,16) were s t r u c t u r a l l y c h a r a c t e r i z e d from t h i s second e x t r a c t . Because LPK was n o t c o l l e c t e d d u r i n g the C - l f r a c t i o n a t i o n s o f the second e x t r a c t , a t h i r d 3000-head e x t r a c t i o n was performed. From t h i s e x t r a c t , LPK was s u c c e s s f u l l y p u r i f i e d , e n z y m a t i c a l l y d e b l o c k e d , and structurally characterized. Mass s p e c t r o m e t r y was n o t a t t e m p t e d (11). A l t h o u g h we f e l t s e c u r e w i t h our s t r u c t u r a l c h a r a c t e r i z a t i o n s o f the s u l f a t e d LSK's, our e v i d e n c e was b a s e d p r i m a r i l y upon the


44 Table


I.

S t r u c t u r e s o f the L e u c o k i n i n s and


Peptide LK-I (Lem-M-I) L K - I I (Lem-M-II) L K - I I I (Lem-M-III) LK-IV (Lem-M-IV) LK-V (Lem-M-V) LK-VI (Lem-M-VI) LK-VII (Lem-M-VII) L K - V I I I (Lem-M-VII)

ACTION

Achetakinins Ref 10 10 14 14 15 15 12 12

Structure DPAFNSWGamide DPGFSSWGamide DQGFNSWGamide DASFHSWGamide GSGFSSWGamide pQSSFHSWGamide DPAFSSWGamide GADFYSWGamide

AK-I (Acd-K-I) SGADFYPWGamide AK-II ( A c d - K - I I ) AYFSPWGamide AK-III (Acd-K-III) ALPFSSWGamide AK-IV (Acd-K-IV) NFKFNPWGamide AK-V (Acd-K-V) AFHSWGamide f Holman, G. M., e t a l . i n Chromatography and I s o l a t i o n o f I n s e c t Hormones and Pheromones. i n p r e s s .

s y n t h e s i s o f s u l f a t e d p e p t i d e s t h a t matched the HPLC r e t e n t i o n times and b i o l o g i c a l a c t i v i t i e s o f the n a t u r a l p r o d u c t s . The LSK's i s o l a t e d from the t h i r d head e x t r a c t p r o v i d e d us w i t h the m a t e r i a l t o o b t a i n a d d i t i o n a l p r o o f o f the s u l f a t e d n a t u r e o f t h e s e peptides. About 1 nmol o f n a t u r a l L S K - I I and an e q u i v a l e n t amount o f s y n t h e t i c L S K - I I were s u b m i t t e d t o f a s t - a t o m bombardment mass s p e c t r o m e t r y a t the USDA Western R e g i o n a l R e s e a r c h C e n t e r a t B e r k e l e y , CA. B o t h compounds showed i o n s a t 1317.2 (MH ) and 1237.3 (MH - S0 ) c o n s i s t e n t w i t h a s i n g l e s u l f a t e group and a p y r o g l u t a m y l r e s i d u e i n the s t r u c t u r e . B a s i c h y d r o l y s i s o f the +

+

3

Table

II.

Structures of S u l f a t e d Insect

Neuropeptides*

Ref. Peptide Structure LSK (Lem-SK-I) 13 EQFEDY ( S 0 H ) GHMRFamide LSK I I ( L e m - S K - I I ) 16 pQSDDY ( S 0 H ) GHMRFamide DSK I (Drm-SK-I) FDDYGHMRFamide 17 DSK I I (Drm-SK-II) GGDDQFDDYGHMRFamide 17 19 PSK (Pea-SK-I) EQFDDY( S 0 H ) GHMRFamide 19 LSK-II ( n o n - s u l f a t e d ) pQSDDYGHMRFamide Lom-SK D O L A S D D Y C SOoH^ GHMRFamide t * S t r u c t u r e s o f DSK I and I I were d e t e r m i n e d by m o l e c u l a r b i o l o g i c a l techniques. E x p r e s s i o n o f t h e s e p e p t i d e s has n o t b e e n demonstrated, f S c h o o f s , L., e t a l . i n Chromatography and I s o l a t i o n o f I n s e c t Hormones and Pheromones. i n p r e s s . 3

3

3

r e m a i n i n g LSK-II n a t u r a l p r o d u c t and a l l o f the LSK n a t u r a l p r o d u c t f o l l o w e d by amino a c i d a n a l y s i s d e m o n s t r a t e d the p r e s e n c e o f a t y r o s i n e s u l f a t e r e s i d u e i n b o t h p e p t i d e s (13,16). LSK and L S K - I I were the f i r s t g a s t r i n / c h o l e c y s t o k i n i n - l i k e p e p t i d e s t o be i s o l a t e d from an i n v e r t e b r a t e s o u r c e .


5.

HOLMANETAL.

Table

III.


Structures

of Leucopyrokinin

(LPK)

45 and R e l a t e d


Peptide Structure LPK (Lem-PK) pOTSFTPRLamide Lom-MT-I GAVPAAQWFSPRLamide Lom-MT-II EGDFTPRLamide Lom-PK DODSGDEWPOOPFVPRLamide Hez-PBAN DPEQIDSRTKYFSPRLamide* Bom-PBAN DPEEMESRTRYFSPRLamide* LPK-5 Ser DOTSFSPRLamide f S c h o o f s , L., e t a l . I n s e c t Biochem.. i n p r e s s . 4 S c h o o f s , L., e t a l . Gen. Comp. E n d o c r i n o l . , i n p r e s s . * Both Hez- and Bom-PBANS' are 3 3 - r e s i d u e p e p t i d e s . Only C - t e r m i n a l 16 r e s i d u e s a r e shown.

Peptides Ref. (11) (21) t

t (22) (23) (20)

the

I n a d d i t i o n t o the L. maderae m y o t r o p i c p e p t i d e s , a s e r i e s o f f i v e m y o t r o p i c p e p t i d e s were i s o l a t e d and s t r u c t u r a l l y c h a r a c t e r i z e d from head e x t r a c t s o f the c r i c k e t , A c h e t a d o m e s t i c u s . w i t h the same p u r i f i c a t i o n system and b i o a s s a y (Holman, G. M., e t a l . Chromatography and I s o l a t i o n o f I n s e c t Hormones and Pheromones. i n press.). The s t r u c t u r e s o f t h e s e f i v e m y o t r o p i c p e p t i d e s , the a c h e t a k i n i n s , are shown i n T a b l e 1. L i k e the l e u c o k i n i n s , the a c h e t a k i n i n s (Ak's) c o n t a i n a C - t e r m i n a l p e n t a p e p t i d e c o r e w h i c h i s r e s p o n s i b l e f o r the m y o t r o p i c a c t i v i t y ( 1 2 ) . I n Ak's I I I and V, the C - t e r m i n a l pentamer i s the same as the l e u c o k i n i n pentamer (Phe-X-Ser-Trp-Gly-NH , where X = Asn, H i s , Ser, o r T y r ) b u t i n Ak's I, I I , and IV a s l i g h t l y d i f f e r e n t pentamer (Phe-X-Pro-Trp-Gly-NH ) i s p r e s e n t . The r e c e p t o r s o f the L. maderae h i n d g u t a r e q u i t e s e n s i t i v e t o the l e u c o k i n i n s (10,12,14,15) and the a c h e t a k i n i n s (Holman, G. M., e t a l . Chromatography and I s o l a t i o n o f I n s e c t Hormones and Pheromones. i n p r e s s . ) as the t h r e s h o l d f o r a c t i v i t y c o n c e n t r a t i o n s range from 0.27 nM t o 0.029 nM. Head t i t e r s o f the l e u c o k i n i n s r a n g e d from 0.48 pmol/head f o r LK-I (10) t o a low o f 0.06 pmol/head f o r LK-VIII (12). Head t i t e r s o f the a c h e t a k i n i n s r a n g e d from 0.15 pmol/head ( A K - I I I ) t o 0.02 pmol/head (AK-II) (Holman, G. M., e t a l . Chromatography and I s o l a t i o n o f I n s e c t Hormones and Pheromones. i n press.). S i n c e the p u b l i c a t i o n i n 1986 (13,16) o f the g a s t r i n / c h o l e c y s t o k i n i n - l i k e l e u c o s u l f a k i n i n s (LSK's), s e v e r a l a d d i t i o n a l i n s e c t s u l f a k i n i n s have been s t r u c t u r a l l y c h a r a c t e r i z e d ( T a b l e I I ) . The s t r u c t u r e s o f the D r o s o p h i l a s u l f a k i n i n s (DSK's) were deduced from a gene sequence which was i s o l a t e d from D r o s o p h i l a genomic DNA and head cDNA l i b r a r i e s (12) • A l t h o u g h e x p r e s s i o n o f the DSK's remains t o be demonstrated, s u l f a t e d s y n t h e t i c r e p l i c a s a r e b i o l o g i c a l l y a c t i v e on the i s o l a t e d c o c k r o a c h h i n d g u t (Holman, u n p u b l i s h e d o b s e r v a t i o n ) as p r e d i c t e d by a s t u d y (18) w h i c h d e m o n s t r a t e d t h a t the hexamer, T y r ( S 0 ) - G l y - H i s - M e t - A r g Phe-NH , was the " c o r e " s t r u c t u r e r e q u i r e d f o r m y o t r o p i c a c t i v i t y . P e r i s u l f a k i n i n , PSK, was i s o l a t e d by chance d u r i n g the i n v e s t i g a t i o n of cardioacceleratory-hypertrehalosaemic peptides of P. a m e r i c a n a t h a t r e s u l t e d i n the s t r u c t u r a l c h a r a c t e r i z a t i o n o f c o r a z o n i n ( 5 ) . Amino a c i d a n a l y s i s and gas-phase s e q u e n c i n g o f an 2

2

3

2



46


ACTION

i n a c t i v e b u t pure HPLC peak y i e l d e d a sequence ( T a b l e I I ) i d e n t i c a l t o LSK e x c e p t f o r a r e s i d u e o f Asp a t p o s i t i o n 4 i n s t e a d o f G l u (19). Barium h y d r o x i d e h y d r o l y s i s f o l l o w e d by amino a c i d a n a l y s i s revealed a s u l f a t e d Tyr residue. S u b s e q u e n t l y , the m y o t r o p i c n a t u r e o f PSK was d e m o n s t r a t e d on the i s o l a t e d h i n d g u t p r e p a r a t i o n o f P. a m e r i c a n a where the t h r e s h o l d o f a c t i v i t y c o n c e n t r a t i o n was d e t e r m i n e d t o be 0.25 nM, v i r t u a l l y the same as the t h r e s h o l d c o n c e n t r a t i o n (0.22 nM) o f LSK on the i s o l a t e d L. maderae h i n d g u t (13). I n a d d i t i o n t o PSK, the n o n - s u l f a t e d form o f L S K - I I was i s o l a t e d and s t r u c t u r a l l y c h a r a c t e r i z e d from the P. a m e r i c a n a c c e x t r a c t s , b u t the b i o l o g i c a l l y a c t i v e s u l f a t e d form was n o t f o u n d (19). The f i n a l p e p t i d e s t r u c t u r e shown i n T a b l e 2 r e p r e s e n t s an i n s e c t s u l f a k i n i n r e c e n t l y i s o l a t e d from b r - c c / c a e x t r a c t s o f the l o c u s t , L o c u s t a m i g r a t o r i a . and s t r u c t u r a l l y c h a r a c t e r i z e d . A m o d i f i c a t i o n o f the f o u r - s t e p HPLC p u r i f i c a t i o n system (10) i n w h i c h a l l g r a d i e n t s were extended t o a h i g h e r f i n a l a c e t o n i t r i l e c o n c e n t r a t i o n was u t i l i z e d . I n a d d i t i o n , a C-8 r e v e r s e - p h a s e column was s u b s t i t u t e d f o r the C-18 column ( S c h o o f s , L., e t a l . Chromatography and I s o l a t i o n o f I n s e c t Hormones and Pheromones. i n press.). The i s o l a t e d h i n d g u t o f L. maderae was u s e d as the bioassay. Lom-SK c o n t a i n s r e s i d u e s o f Leu and A l a a t the 2- and 3 - p o s i t i o n s , r e s p e c t i v e l y . The remainder o f the sequence i s i d e n t i c a l w i t h the 2-10 sequence o f L S K - I I . The p r e s e n c e o f s i m i l a r i n s e c t s u l f a k i n i n s i n s p e c i e s as d i v e r s e as c o c k r o a c h e s and D r o s o p h i l a s u g g e s t s t h i s p e p t i d e f a m i l y may be w i d e l y d i s t r i b u t e d among the I n s e c t a . I n a d d i t i o n , the C - t e r m i n a l o c t a p e p t i d e c o r e appears t o have been c o n s e r v e d d u r i n g evolution. The o c t a p e p t i d e l e u c o p y r o k i n i n (LPK) was the e l e v e n t h and f i n a l m y o t r o p i c p e p t i d e i s o l a t e d from L. maderae ( 1 1 ) . U n l i k e the l e u c o k i n i n s and LSK's, LPK e x h i b i t s a m y o t r o p i c a c t i v i t y on the m u s c l e s o f the c o c k r o a c h f o r e g u t and o v i d u c t (Holman, G. M. and Nachman, R. J . , u n p u b l i s h e d o b s e r v a t i o n . ) . LPK was the h i g h e s t t i t e r e d o f the Leucophaea m y o t r o p i n s (1.36 pmol/head) b u t was the l e a s t p o t e n t on the i s o l a t e d h i n d g u t p r e p a r a t i o n ( t h r e s h o l d c o n c e n t r a t i o n =0.6 nM). LPK s h a r e s a 50% sequence i d e n t i t y w i t h Pea-HTH-II a t the 1, 4, 5, and 6 p o s i t i o n s and i s a m i d a t e d a t the C - t e r m i n u s . Y e t LPK does n o t c o n t a i n T r p , which i s p r e s e n t a t p o s i t i o n 8 i n e v e r y AKH/RPCH p e p t i d e . F u r t h e r m o r e , LPK i s p o s i t i v e l y c h a r g e d w i t h a r e s i d u e o f A r g a t p o s i t i o n 8. These s t r u c t u r a l o b s e r v a t i o n s show t h a t LPK i s n o t a member o f the AKH/RPCH f a m i l y . A s t u d y o f LPK a n a l o g s s u p p o r t s t h i s v i e w ( 2 0 ) . LPK a n a l o g s i n w h i c h Asn r e p l a c e d A r g a t p o s i t i o n 7 o r T r p r e p l a c e d Leu a t p o s i t i o n 8 e x h i b i t e d d r a s t i c a l l y r e d u c e d ( 1 0 0 0 - f o l d ) myotropic a c t i v i t y . F i n a l l y , N - t e r m i n a l t r u n c a t i o n o f AKH/RPCH p e p t i d e s d e s t r o y s b i o l o g i c a l a c t i v i t y whereas LPK can be t r u n c a t e d t o the C - t e r m i n a l p e n t a p e p t i d e c o r e (Phe-Thr-Pro-Arg-Leu-NH ) and s t i l l r e t a i n s i g n i f i c a n t (25%) m y o t r o p i c a c t i v i t y ( 2 0 ) . A l t h o u g h LPK was the o n l y p e p t i d e i s o l a t e d from the Leucophaea head e x t r a c t s t h a t c o n t a i n e d the amidated C - t e r m i n a l pentapeptide, Phe-X-Pro-Arg-Leu-NH (X = T h r ) , a d d i t i o n a l n e u r o p e p t i d e s c o n t a i n i n g t h a t p e n t a p e p t i d e c o r e have s u b s e q u e n t l y been i s o l a t e d from o t h e r i n s e c t s p e c i e s and s t r u c t u r a l l y c h a r a c t e r i z e d ( T a b l e 2

2


5.

HOLMAN ETAL.

47


III). Lom-MT-I (X - S e r ) ( 2 1 ) , Lom-MT-II (X - T h r ) ( S c h o o f s , L., e t a l . I n s e c t Biochem.. i n p r e s s . ) , and Lom-PK (X - V a l ) ( S c h o o f s , L., e t a l . Gen. Comp. E n d o c r i n o l . , i n p r e s s . ) were i s o l a t e d from br-cc/ca extracts o f Locusta migratoria. The i s o l a t e d h i n d g u t p r e p a r a t i o n o f Leucophaea was u s e d as t h e b i o a s s a y . None o f t h e t h r e e l o c u s t n e u r o p e p t i d e s appear t o s t i m u l a t e c o n t r a c t i o n s o f t h e i s o l a t e d l o c u s t hindgut. However, b o t h Lom-MT's s t i m u l a t e c o n t r a c t i o n s o f t h e i s o l a t e d l o c u s t o v i d u c t w h i l e Lom-PK s t i m u l a t e s the i s o l a t e d l o c u s t f o r e g u t . Two r e c e n t l y c h a r a c t e r i z e d n e u r o p e p t i d e s , t h e pheromone b i o s y n t h e s i s a c t i v a t i n g neurohormones (PBAN's) b o t h c o n t a i n t h e C - t e r m i n a l pentamer P h e - S e r - P r o - A r g Leu-NH . The two l e p i d o p t e r a n n e u r o p e p t i d e s b o t h c o n t a i n 33 amino a c i d r e s i d u e s , o f which o n l y s i x d i f f e r . Hez-PBAN was i s o l a t e d from the a d u l t c o t t o n b o l l worm, H e l i o t h i s z e a (22) w h i l e Bom-PBAN was i s o l a t e d from t h e silkworm, Bombyx m o r i ( 2 3 ) . L i k e LPK, Bom-PBAN can t o l e r a t e N - t e r m i n a l t r u n c a t i o n . The amidated C - t e r m i n a l d e c a p e p t i d e o f Bom-PBAN r e t a i n e d a r e d u c e d b u t d e f i n i t e PBA a c t i v i t y (23). Based upon t h e o b s e r v a t i o n s o f Nachman e t a l . (20), we p r e d i c t e d t h a t t h e PBAN's would s t i m u l a t e t h e i s o l a t e d v i s c e r a l m u s c l e s o f Leucophaea. T h i s p r e d i c t i o n was c o r r e c t . Hez-PBAN ( N l e , N l e ) s t i m u l a t e d the c o n t r a c t i o n s o f the i s o l a t e d Leucophaea h i n d g u t above a t h r e s h o l d c o n c e n t r a t i o n o f 14.5 ± 4.2 nM (Holman, G. M. and Nachman, R. J . , u n p u b l i s h e d . ) . At threshold c o n c e n t r a t i o n , Hez-PBAN ( N l e , N l e u ) was about f o u r - f o l d l e s s a c t i v e t h a n LPK on t h e i s o l a t e d Leucophaea o v i d u c t (6.3 ± 2.5 nM v s . 1.6 ± 0.26 nM, r e s p e c t i v e l y ) (Holman, G. M. and Nachman, R. J . , unpublished.). The s t u d i e s above s u g g e s t t h a t t h e LPK f a m i l y o f p e p t i d e s may be w i d e l y d i s t r i b u t e d among t h e i n s e c t s p e c i e s and p o s s e s s a number o f f u n c t i o n s . The p e n t a p e p t i d e c o r e , Phe-X-Pro-Arg-Leu-NH , appears t o have been w e l l c o n s e r v e d d u r i n g evolution.


2

5

u

5

1 A

2

Neuropeptide I n h i b i t o r s o f V i s c e r a l Muscle O n l y two n e u r o p e p t i d e s t h a t i n h i b i t t h e spontaneous c o n t r a c t i o n s o f v i s c e r a l muscle have been s t r u c t u r a l l y c h a r a c t e r i z e d . These two p e p t i d e s , which c o n t a i n a C - t e r m i n a l FLRFamide t e t r a m e r a r e a l m o s t i d e n t i c a l as shown: Leucomyosuppressin SchistoFLRFamide

pQDVDHVFLRFamide

(24)

PDVDHVFLRFamide

(25)

L e u c o m y o s u p p r e s s i n (LMS) was i s o l a t e d c o n c u r r e n t l y w i t h t h e l e u c o k i n i n s , l e u c o p y r o k i n i n s , and l e u c o s u l f a k i n i n s from Leucophaea head e x t r a c t s (24). IMS r e v e r s i b l y i n h i b i t s t h e spontaneous c o n t r a c t i o n s o f t h e i s o l a t e d Leucophaea h i n d g u t i n a dose-dependent fashion. The t h r e s h o l d o f a c t i v i t y c o n c e n t r a t i o n was 0.078 nM, and each c o c k r o a c h head c o n t a i n e d 0.23 pmol IMS ( 2 4 ) . A s i m i l a r p e p t i d e , S c h i s t o F L R F a m i d e , was r e c e n t l y i s o l a t e d from e x t r a c t s o f the l o c u s t ( S c h i s t o c e r c a g r e g a r i a ) t h o r a c i c nervous system by g e l f i l t r a t i o n f o l l o w e d by RP-HPLC on a C-18 column. Samples were i n i t i a l l y f r a c t i o n a t e d w i t h a w a t e r : a c e t o n i t r i l e g r a d i e n t u s i n g h e p t a f l u o r o b u t y r i c a c i d as t h e i o n - p a i r i n g agent.

American Chemical Society Library 1155 16th St., H.W. Washington, D.CNeuropeptides 20036 Menn et al.; Insect ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


48


ACTION

The a p p r o p r i a t e f r a c t i o n was c o l l e c t e d and r e c h r o m a t o g r a p h e d on the same column w i t h the same g r a d i e n t b u t a d i f f e r e n t i o n - p a i r i n g r e a g e n t (10 mM t r i e t h y l a m i n e a d j u s t e d t o pH 6.5 w i t h t r i f l u o r o a c e t i c acid). S c h i s t o F L R F a m i d e was i s o l a t e d on the b a s i s o f c r o s s - r e a c t i v i t y t o a n t i b o d i e s r a i s e d a g a i n s t the m o l l u s c a n p e p t i d e FMRFamide. Sequence a n a l y s i s was p e r f o r m e d w i t h a p u l s e d - l i q u i d phase sequencer u s i n g Edman c h e m i s t r y ( 2 5 ) . L i k e LMS, S c h i s t o F L R F a m i d e i s a p o t e n t i n h i b i t o r o f v i s c e r a l muscle c o n t r a c t i o n . When a p p l i e d to the s e m i - i s o l a t e d S c h i s t o c e r c a h e a r t p r e p a r a t i o n , c o n c e n t r a t i o n s o f SchistoFLRMamide between 1 nM and 0.1 nM p r o d u c e d an o b s e r v a b l e i n h i b i t o r y e f f e c t . A t 1000 nM, spontaneous h e a r t c o n t r a c t i o n s were a b o l i s h e d and remained so f o r s e v e r a l minutes a f t e r removal o f the p e p t i d e - c o n t a i n i n g s o l u t i o n (24). I n a d d i t i o n , S c h i s t o F L R F a m i d e had an e f f e c t upon the l o c u s t e x t e n s o r - t i b i a e muscle p r e p a r a t i o n ( s o m a t i c m u s c l e ) . Low c o n c e n t r a t i o n s (.01 nM-10 nM) p r o d u c e d a p o t e n t i a t i o n o f the a m p l i t u d e o f slow motor n e u r o n i n d u c e d t w i t c h t e n s i o n w h i l e h i g h e r c o n c e n t r a t i o n s (100 nM-1000 nM) p r o d u c e d a v a r i a b l e b i p h a s i c r e s p o n s e w i t h an a m p l i t u d e o f c o n t r a c t i o n d e c r e a s e f o l l o w e d by an i n c r e a s e (25). LMS was i s o l a t e d on the b a s i s o f i t s a c t i o n upon v i s c e r a l muscle. However, i t a l s o e f f e c t s motor neurons ( 2 6 ) . LMS a t t e n u a t e d the evoked t r a n s m i t t e r r e l e a s e from the p r e s y n a p t i c membrane o f e x c i t a t o r y motor neurons t e r m i n a t i n g on the s k e l e t a l muscle o f the mealworm, T e n e b r i o m o l i t o r , b u t had no p o s t s y n a p t i c e f f e c t s on t h a t p r e p a r a t i o n . A l t h o u g h the mechanisms o f LMS-induced i n h i b i t i o n o f e x c i t a t o r y p r e s y n a p t i c p o t e n t i a l s have n o t b e e n p r e c i s e l y i d e n t i f i e d , one p r e l i m i n a r y experiment s u g g e s t e d t h a t m e t a b o l i t e s o f a r a c h i d o n i c a c i d may f u n c t i o n as a s e c o n d messenger f o r LMS a t t h a t s i t e ( 2 6 ) . Conclusions

and

Future

Directions

B e g i n n i n g w i t h the i n i t i a l s u c c e s s o f Brown and S t a r r a t t (7,8) and c o n t i n u i n g t o the p r e s e n t , m y o t r o p i c b i o a s s a y s have p l a y e d a major r o l e i n the i s o l a t i o n and s t r u c t u r a l c h a r a c t e r i z a t i o n o f i n s e c t neuropeptides. The s u c c e s s w i l l l i k e l y c o n t i n u e s i n c e m y o t r o p i c b i o a s s a y s a r e s e n s i t i v e , r a p i d , r e l i a b l e , and r e p r o d u c i b l e ; c h a r a c t e r i s t i c s d e s i r a b l e t o the i s o l a t i o n s p e c i a l i s t . Insect n e u r o p e p t i d e i s o l a t i o n p r o j e c t s u t i l i z i n g a m y o t r o p i c b i o a s s a y have been r e s p o n s i b l e f o r the d i s c o v e r y o f f o u r s t r u c t u r a l l y u n i q u e p e p t i d e f a m i l i e s and the i n i t i a l d e m o n s t r a t i o n o f the e x i s t e n c e o f n a t u r a l - a n a l o g s e r i e s which may be q u i t e common i n i n s e c t s . A d d i t i o n a l m y o t r o p i c / i n h i b i t o r y n e u r o p e p t i d e s t r u c t u r e s w i l l be c h a r a c t e r i z e d on the b a s i s o f i m m u n o l o g i c a l s i m i l a r i t y to v e r t e b r a t e and i n v e r t e b r a t e p e p t i d e s t r u c t u r e s , and a l s o w i t h the t e c h n i q u e s o f molecular biology. Two i n i t i a l s u c c e s s e s w i t h t h o s e methods a r e d i s c u s s e d i n t h i s r e p o r t (17,25). In a d d i t i o n , a n t i b o d i e s r a i s e d a g a i n s t FMRFamide were u s e d to i s o l a t e and s t r u c t u r a l l y c h a r a c t e r i z e a n o n a p e p t i d e c o n t a i n i n g C - t e r m i n a l FMRFamide from head e x t r a c t s o f D r o s o p h i l a (26). S u b s e q u e n t l y , the gene t h a t codes f o r t h i s n o n a p e p t i d e and e i g h t o t h e r FMRFamide-related s t r u c t u r e s was i s o l a t e d and sequenced (27,28).



5.

HOLMAN ETAL.


49

As new i n s e c t n e u r o p e p t i d e s t r u c t u r e s a r e r e v e a l e d , s y n t h e t i c r e p l i c a s w i l l become a v a i l a b l e . S t u d i e s o f t h e s e s y n t h e t i c m a t e r i a l s w i l l advance o u r u n d e r s t a n d i n g o f c h e m i c a l messengers and the f u n c t i o n s they c o n t r o l . Most l i k e l y , new p h y s i o l o g i c a l and biochemical a c t i v i t i e s i n f l u e n c e d by m u l t i f u n c t i o n a l peptides w i l l be d i s c o v e r e d . E v o l u t i o n a r y r e l a t i o n s h i p s among i n s e c t s p e c i e s and between i n s e c t s and o t h e r a n i m a l s , b o t h v e r t e b r a t e and i n v e r t e b r a t e , w i l l be c l a r i f i e d . Immunocytochemical s t u d i e s w i l l r e v e a l s p e c i f i c i d e n t i f i a b l e neurons t h a t s y n t h e s i z e t h e s e p e p t i d e s , and b i o c h e m i c a l s t u d i e s w i l l demonstrate t h e p r e c i s e mechanisms o f p e p t i d e biosynthesis, processing, p o s t - t r a n s l a t i o n a l modification, s e c r e t i o n , t r a n s p o r t , and metabolism. S t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s and computer m o d e l i n g s t u d i e s w i l l demonstrate how b i o l o g i c a l i n f o r m a t i o n i s encoded upon t h e s t r u c t u r e o f t h e s e p e p t i d e s and w i l l i n c r e a s e o u r knowledge o f m e s s e n g e r - r e c e p t o r interactions. Insect neuropeptides, i n c l u d i n g the myotropins, a r e the master r e g u l a t o r s o f i n s e c t development, metabolism, h o m e o s t a s i s , b e h a v i o r , and r e p r o d u c t i o n . They a r e t h e m o l e c u l a r messengers t h a t p r o v i d e c o o r d i n a t i o n between t h e t i s s u e s t h a t make up t h e organisms we c a l l i n s e c t s .

Literature Cited 1. Holman, G. M.; Cook, B. J. J. Insect Physiol. 1970, 16, 1891-1907. 2. Witten, J. L.; Schaffer, M. H.; O'Shea, M.; Cook, J. C.; Hemling, M. E.; Rinehart, K. L. Biochem. Biophys. Res. Commun. 1984, 124, 350-358. 3. Scarborough, R. M.; Jamieson, G. C.; Kalish, F.; Kramer, S. J.; McEnroe, G. A.; Miller, C. A.; Schooley, D. A. Proc. Natl. Acad. Sci. USA 1984, 81, 5575-5579. 4. O'Shea, M.; Witten, J. L.; Schaffer, M. H. J. Neurosci. 1984, 4, 521-529. 5. Veenstra, J. A. FEBS Lett. 1989, 250, 231-234. 6. Brown, B. E. Science 1967, 155, 595-597. 7. Brown, B. E.; Starratt, A. N. J. Insect Physiol. 1975, 21, 1879-1881. 8. Starratt, A. N.; Brown, B. E. Life Sci. 1975, 17, 1253-1256. 9. Orchard, I.; Belanger, J. H.; Lange, A. B. J. Neurobiol. 1989, 20, 470-496. 10. Holman, G. M.; Cook, B. J . ; Nachman, R. J. Comp. Biochem. Physiol. 1986, 84C, 205-211. 11. Holman, G. M.; Cook, B. J.; Nachman, R. J. Comp. Biochem. Physiol. 1986, 85C, 219-224. 12. Holman, G. M.; Cook, B. J.; Nachman, R. J. Comp. Biochem. Physiol. 1987, 88C, 31-34. 13. Nachman, R. J.; Holman, G. M.; Haddon, W. F.; Ling, N. Science 1986, 234, 71-73. 14. Holman, G. M.; Cook, B. J.; Nachman, R. J. Comp. Biochem. Physiol. 1986, 84C, 271-276. 15. Holman, G. M.; Cook, B. J.; Nachman, R. J. Comp. Biochem. Physiol. 1987, 88C, 27-30. 16. Nachman, R. J.; Holman, G. M.; Cook, B. J.; Haddon, W. F.; Ling, N. Biochem. Biophys. Res. Commun. 1986, 140, 357-364.



50

INSECT NEUROPEPTIDES: CHEMISTRY, BIOLOGY, AND ACTION

17. Nichols, R.; Schneuwly, S. A.; Dixon, J. E. J. Biol. Chem. 1988, 263, 12167-12170. 18. Nachman, R. J.; Holman, G. M.; Haddon, W. F.; Hayes, T. K. Pept. Res. 1989, 2, 171-177. 19. Veenstra, J. A. Neuropeptides 1989, 14, 145-149. 20. Nachman, R. J.; Holman, G. M.; Cook, B. J. Biochem. Biophys. Res. Commun. 1986, 137, 936-942. 21. Schoofs, L.; Holman, G. M.; Hayes, T. K.; Tips, A.; Nachman, R. J.; Vandesande, E.; DeLoof, A. Peptides 1990, 11, 427-433. 22. Raina, A. K.; Jaffe, H.; Kempe, T. G.; Keim, P.; Blacher, R. W.; Fales, H. M.; Riley, C. T.; Klun, J. A.; Ridgway, R. L.; Hayes, D. K. Science 1989, 244, 796-798. 23. Kitamura, A.; Nagasawa, H.; Kataoka, H.; Inoue, T.; Matsumoto, S.; Ando, T.; Suzuki, A. Biochem. Biophys. Res. Commun. 1989, 163, 520-526. 24. Holman, G. M.; Cook, B. J . ; Nachman, R. J. Comp. Biochem. Physiol. 1986, 85C, 329-333. 25. Robb, S.; Packman, L. C.; Evans, P. D. Biochem. Biophys. Res. Commun. 1989, 160, 850-856. 26. Yamamoto, D.; Ishikawa, S.; Holman, G. M.; Nachman, R. J. Neurosci. Lett. 1988, 95, 137-142. 27. Nambu, J. R.; Murphy-Erdosh, C.; Andrews, P. C.; Feistner, G. J.; Scheller, R. H. Neuron 1988, 1, 55-61. 28. Schneider, L. E.; Taghert, P. H. Proc. Natl. Acad. Sci. USA 1988, 85, 1993-1997. RECEIVED September 7, 1990


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