Insect Cuticle Tanning - ACS Symposium Series (ACS Publications)

Chapter 7 Insect Cuticle Tanning Enzymes and Cross-Link Structure K.

J. Kramer , T. D. Morgan , T. L Hopkins , Allyson Christensen , 1

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and Jacob Schaefer Downloaded by PENNSYLVANIA STATE UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: January 9, 1991 | doi: 10.1021/bk-1991-0449.ch007

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U.S. Grain Marketing Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Manhattan, KS 66502 Department of Entomology, Kansas State University, Manhattan, KS 66506 Department of Chemistry, Washington University, St Louis, MO 63130 1

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Insects periodically secrete and stabilize a cuticular exoskeleton to allow for growth and differentiation during development to the adult stage. Sclerotization or tanning is a vital process in which specific regions of the newly secreted cuticle are stabilized by the formation of cross-links between biopolymers such as protein and chitin. Solid state NMR analysis of pupal cuticle has detected covalent bonding between aromatic or aliphatic carbons of catecholamines and protein nitrogen. Weaker secondary bonding and dehydration may also occur as phenolic content increases. The tanning agents are electrophilic deriv atives (o-quinones and ρ-quinone methides or free radicals) of catecholamines that may be involved in both sclerotization and pigmentation. Phenolox idases such as laccases and tyrosinases and other types of enzymes such as isomerases catalyze the formation of reactive tanning agents from N-acylcate cholamines. Understanding the chemistry of insect cuticular sclerotization could lead to the development of new insecticides. I n s e c t s a n d o t h e r a r t h r o p o d s o b t a i n s t r u c t u r a l s u p p o r t and p r o t e c t i o n from a c u t i c u l a r e x o s k e l e t o n s e c r e t e d by a s i n g l e l a y e r o f epidermal c e l l s . The s t a g e s o f growth and development are d e l i n e a t e d b y d e p o s i t i o n , e x p a n s i o n , s t a b i l i z a t i o n and p i g m e n t a t i o n o f the new c u t i c l e and shedding o f t h e o l d c u t i c l e . Cuticular sclerotization o c c u r s b o t h b e f o r e and a f t e r e c d y s i s i n g e n e t i c a l l y determined p a t t e r n s . The e x o s k e l e t o n may be expandable t o accommo date c o n t i n u e d growth, f l e x i b l e t o a l l o w a r t i c u l a t i o n between j o i n t s and segments, o r r i g i d t o p r o v i d e m e c h a n i c a l s t a b i l i t y and r e s i s t a n c e t o compression. The p h y s i c a l and c h e m i c a l p r o p e r t i e s o f t h i s 0097-6156/91/0449-0087$06.00A) © 1991 American Chemical Society In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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NATURALLY OCCURRING PEST BIOREGULATORS

multifunctional s t r u c t u r e depend on the types and amounts o f minerals, lipids, c h i t i n , p h e n o l s , s t r u c t u r a l p r o t e i n s and enzymes t h a t c a t a l y z e the assembly and s t a b i l i z a t i o n o f the completed exoskeleton. S c l e r o t i z a t i o n i s a complex c h e m i c a l p r o c e s s i n v o l v i n g o x i d a t i o n of diphenols t o q u i n o n o i d d e r i v a t i v e s and p o s s i b l y f r e e r a d i c a l s t h a t form c o v a l e n t bonds between macromolecules i n the c u t i c u l a r matrix. C r o s s - l i n k s and o t h e r types o f bonds produce a h i g h l y i n s o l u b l e d e h y d r a t e d s t r u c t u r e r e s i s t a n t t o c h e m i c a l and p h y s i c a l degradation. Enzymes w h i c h c a t a l y z e the o x i d a t i o n and isomerization of phenolic metabolites i n the c u t i c l e i n c l u d e p h e n o l o x i d a s e s , p e r o x i dases, and i s o m e r a s e s . Dopamine, N-acetyldopamine and N-£-alanyldopamine are i m p o r t a n t s u b s t r a t e s f o r these enzymes and are t r a n s ported from the e p i d e r m i s i n t o newly s e c r e t e d c u t i c l e p r i o r to s c l e r o t i z a t i o n and p i g m e n t a t i o n (1,2). Dopamine i s a s s o c i a t e d w i t h the synthesis of b l a c k m e l a n i n i n c u t i c l e , and N-£-alanyldopamine w i t h the s y n t h e s i s o f brown s c l e r o t i n . N-Acetyldopamine i s o f t e n a s s o c i a t e d w i t h the s y n t h e s i s o f c o l o r l e s s s c l e r o t i n , a l t h o u g h the presence o f dopamine o r N-/9-alanyldopamine i n s t r u c t u r e s c o n t a i n i n g N-acetyldopamine w i l l a l s o r e s u l t i n c o l o r development. The o x i d i z e d p r o d u c t s t h a t can be formed from t h e s e c a t e c h o l a m i n e s include o-quinones and t h e i r isomeric p-quinone methides and a,0-dehydrocatecholamines, as well as semiquinones and their isomeric carbon r a d i c a l s (3,4). Figure 1 i l l u s t r a t e s a possible two-electron o x i d a t i v e pathway f o r c o n v e r s i o n o f d i p h e n o l s t o some o f t h e s e p r o d u c t s and includes metabolites involved i n c u t i c u l a r melanization. The reactions i n c l u d e two e l e c t r o n o x i d a t i o n to o-quinones ( F i g . 1, compound 2 ) , spontaneous c y c l i z a t i o n t o l e u c o aminochromes ( 3 ) , t w o - e l e c t r o n o x i d a t i o n t o p-quinoneimines ( 5 ) , i n d o l i z a t i o n to 5,6-dihydroxyindole d e r i v a t i v e s (6), two-electron o x i d a t i o n to i n d o l e - 5 , 6 - q u i n o n e s ( 7 ) , and p o l y m e r i z a t i o n t o melanochromes and m e l a n i n s . Insects u t i l i z e N-acylated catecholamines f o r s c l e r o t i z a t i o n because the amino group i s b l o c k e d from i n t r a m o l e c u l a r c y c l i z a t i o n and subsequent m e l a n i n f o r m a t i o n . The o-quinones are s u f f i c i e n t l y l o n g - l i v e d so t h a t they may r e a c t w i t h n u c l e o p h i l i c groups i n the c u t i c l e o r they may t a u t o m e r i z e t o r e a c t i v e p-quinone methides (Fig. 1, compound 4 and F i g . 2, compound 5) f o r s i d e c h a i n c r o s s l i n k formation. I t i s p o s s i b l e t h a t the p-quinone methide may isomerize t o an a,0-dehydrocatecholamine ( F i g . 1, compound 8 and Fig. 2, compound 6) w h i c h may be s u b s e q u e n t l y o x i d i z e d t o an α,/3-dehydro-o-quinone or t o o t h e r p r o d u c t s ( F i g . 2, compound 9). The r e s u l t i n g e l e c t r o p h i l i c α and β c a r b o n may be i n volved i n c r o s s - l i n k formation. I t s h o u l d be n o t e d t h a t , i n a d d i t i o n t o two e l e c t r o n o x i d a t i o n , one e l e c t r o n o x i d a t i v e p r o d u c t s such as semiquinones (2) may be produced i n c u t i c l e e i t h e r e n z y m a t i c a l l y by laccases and p e r o x i d a s e s o r s p o n t a n e o u s l y by d i s p r o p o r t i o n a t i o n o f the o-quinone and diphenol. The semiquinones may i s o m e r i z e to c a r b o n r a d i c a l s ( 3 ) , w h i c h may undergo a d d i t i o n s , s u b s t i t u t i o n s , a b s t r a c t i o n s and isomerizations to a l a r g e number o f p o s s i b l e products. The complex m i x t u r e o f o x i d a t i o n p r o d u c t s from N - a c y l c a t e cholamines w i l l be discussed h e r e , t o g e t h e r w i t h the e v i d e n c e f o r d i p h e n o l - p r o t e i n adducts and c r o s s - l i n k s i n s c l e r o t i z e d c u t i c l e .

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


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CUTICULAR OXIDASES


The p h e n o l o x i d a s e s a r e a r e l a t e d group o f copper c o n t a i n i n g enzymes t h a t c a t a l y z e the o x i d a t i o n o f phenols t o quinones i n a n i m a l s and plants (5,6). Two d i s t i n c t types o f p h e n o l o x i d a s e s t h a t have substrate specificities and i n h i b i t o r s e n s i t i v i t i e s resembling t y p i c a l t y r o s i n a s e s and l a c c a s e s a r e found i n d i f f e r e n t types o f i n s e c t c u t i c l e (2,8). P e r o x i d a s e s , heme-containing enzymes t h a t a l s o o x i d i z e d i p h e n o l s t o quinones, may be p r e s e n t i n c u t i c l e and may p l a y a r o l e i n s c l e r o t i z a t i o n (9,10). Tyrosinase. T y r o s i n a s e i s a m u l t i - f u n c t i o n a l copper o x i d a s e t h a t a c t s b o t h as a monophenol monooxygenase (EC 1.14.18.1) and as a catechol oxidase (EC 1.10.3.1, o - d i p h e n o l O2 o x i d o r e d u c t a s e ) . Most o f t h e monophenol monooxygenase a c t i v i t y o f the p u p a l i n t e g u ment c o n t a i n i n g t y r o s i n a s e appears t o be l o c a t e d i n the e p i d e r m i s i n s t e a d o f the c u t i c l e (11,12, Morgan et.al. u n p u b l i s h e d d a t a ) . T h i s l o c a l i z a t i o n suggests t h a t the p r i m a r y r o l e o f t y r o s i n a s e may be i n t h e e p i d e r m i s and t h a t some o t h e r type o f p h e n o l o x i d a s e may be present i n the pupal c u t i c l e . Studies with other insect species have shown t h a t t y r o s i n a s e i s found t y p i c a l l y i n f l e x i b l e l a r v a l c u t i c l e , where i t may be i n v o l v e d i n r e p a i r o f wounds i n s o f t cuticle (8,13,14). Tyrosinase i s probably present i n f l e x i b l e , c o l o r l e s s c u t i c l e as an i n a c t i v e proenzyme t h a t can be a c t i v a t e d by a p r o t e a s e f o l l o w i n g wounding. A c t i v e p h e n o l o x i d a s e i s p r e s e n t i n r e g i o n s o f t h e l a r v a l c u t i c l e t h a t m e l a n i z e . I n the b l o w f l y , Lucilia cuprina, active tyrosinase i s present i n e p i c u t i c u l a r f i l aments and p r o t y r o s i n a s e i s a p p a r e n t l y p r e s e n t i n t h e p r o c u t i c l e o f the l a r v a e (15) . The p h e n o l o x i d a s e t h a t i s r e s p o n s i b l e f o r the synt h e s i s o f m e l a n i n from dopamine i n l a r v a l c u t i c l e o f M. sexta has been p u r i f i e d , b u t i t has n o t been r i g o r o u s l y t e s t e d f o r monop h e n o l o x i d a s e a c t i v i t y (16). Laccase. Laccase (EC 1.10.3.2, d i p h e n o l : O2-oxidoreductase) i s a c o p p e r - c o n t a i n i n g p h e n o l o x i d a s e t h a t has been d e t e c t e d i n c u t i c l e s o f a t l e a s t 16 s p e c i e s o f i n s e c t s , i n c l u d i n g t h e p u p a l c u t i c l e o f M. sexta (12,18, Thomas et.al. unpublished data). I t i s present i n the i n n e r e p i c u t i c l e o f L. cuprina (JL5) · T h i s enzyme i s p r e s e n t i n t h e a c t i v e form d u r i n g s c l e r o t i z a t i o n and i t p r o b a b l y generates the o-quinones that participate i n cross-linking reactions. A common problem encountered i n s t u d i e s o f c u t i c u l a r laccase i s i t s i n s o l u b i l i t y . S o l u b l e l a c c a s e s have been e x t r a c t e d from l a r v a l d i p t e r a n c u t i c l e , b u t endogenous p r o t e a s e s a r e a l s o known t o be p r e s e n t i n such c u t i c l e . Yamazaki (19) was the f i r s t t o d i s c o v e r l a c c a s e s i n i n s e c t s . She has r e c e n t l y used sodium d o d e c y l s u l f a t e t o e x t r a c t l a c c a s e s from Bombyx mori c u t i c l e . The enzymes can be i d e n t i f i e d by immunoblotting t e c h n i q u e s a f t e r s e p a r a t i o n by sodium d o d e c y l s u l f a t e p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s , b u t the enzymatic a c t i v i t y has n o t y e t been r e c o v e r e d a f t e r the d e t e r g e n t t r e a t m e n t ( 2 0 ) . The standard p r o t o c o l i n v o l v i n g p r o t e o l y s i s w i t h exogenous t r y p s i n s o l u b i l i z e s l a c c a s e from the p u p a l c u t i c l e o f M. sexta (IS). Following incubation of pupal cuticle from M. sexta with



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c h y m o t r y p s i n , we found t h a t l a c c a s e a c t i v i t y i n the s u p e r n a t a n t i s e l e v a t e d a f t e r t r e a t m e n t w i t h t r y p s i n (Morgan et.al. unpublished data). In a d d i t i o n to i t s e f f e c t i v e n e s s i n s o l u b i l i z i n g laccase, t r y p s i n appears t o be an a c t i v a t o r o f p r o l a c c a s e . Yamazaki (20.21) has r e p o r t e d a proenzyme from the c u t i c l e o f B. mori t h a t may be s o l u b i l i z e d by t r e a t m e n t w i t h c h y m o t r y p s i n . However, Andersen (22) and B a r r e t t (23) have not o b t a i n e d evidence f o r a p r o l a c c a s e i n t h e i r e x t e n s i v e work on c u t i c u l a r p h e n o l o x i d a s e s . The c a t a l y t i c p r o p e r t i e s o f l a c c a s e d i f f e r from t y r o s i n a s e i n t h a t l a c c a s e c a t a l y s e s the o x i d a t i o n o f d i p h e n o l s i n discrete one-electron s t e p s , whereas a s i n g l e t w o - e l e c t r o n s t e p occurs w i t h tyrosinase (5,6). U n l i k e t y r o s i n a s e , l a c c a s e has no monophenol o-hydroxylating activity. Both o- and ρ-diphenols are o x i d i z e d by l a c c a s e , but t y r o s i n a s e o x i d i z e s o n l y the former compounds. Methylhydroquinone is a ρ-diphenol that i s o f t e n used to estimate l a c c a s e a c t i v i t y because o f the r e l a t i v e l y h i g h v e l o c i t y o b t a i n e d w i t h t h i s s u b s t r a t e . The h i g h r a t e o f enzymatic o x i d a t i o n may be due, i n p a r t , to the low o x i d a t i o n p o t e n t i a l o f methylhydro quinone and t h i s property can e a s i l y l e a d to e x p e r i m e n t a l e r r o r s . For example, Ν-β-alanyldopamine quinone is an effective o x i d i z i n g agent f o r methylhydroquinone; t h e r e f o r e , a d d i t i o n o f t r a c e amounts o f N-£-alanyldopamine causes r a p i d o x i d a t i o n o f methyl hydroquinone in the presence o f t y r o s i n a s e (Morgan et. al. unpublished data). N o n e t h e l e s s , methylhydroquinone i s a good sub s t r a t e f o r the i n s o l u b l e r e s i d u e o f u n s c l e r o t i z e d p u p a l c u t i c l e and the p a r t i a l l y p u r i f i e d t r y p s i n - s o l u b i l i z e d l a c c a s e from M. sexta (18, Morgan et. al. u n p u b l i s h e d d a t a ) . Although the trypsin-solubilized laccase from M. sexta o x i d i z e s N-0-alanyldopamine to i t s o-quinone, the o-quinone does not accumulate to a high concentration (18, Morgan et. al. unpublished data). I n s t e a d , an enzyme i n the p r e p a r a t i o n , p o s s i b l y the l a c c a s e i t s e l f , appears t o c a t a l y z e the c o n v e r s i o n o f the o-quinone to a p-quinone methide or another i n t e r m e d i a t e which reacts with water to y i e l d the 0 - h y d r o x y l a t e d d e r i v a t i v e o f Ν-/3-alanylnor e p i n e p h r i n e . F u r t h e r s t u d i e s o f p u r i f i e d M. sexta l a c c a s e are needed t o determine whether i t i s a b i f u n c t i o n a l enzyme w h i c h promotes not o n l y ο-diphenol o x i d a t i o n , but a l s o isome r i z a t i o n o f the o-quinone t o a p-quinone methide. U n l i k e the laccase preparation, t y r o s i n a s e from M. sexta has l i t t l e e f f e c t on the r a t e o f 0 - h y d r o x y l a t i o n . The f u n c t i o n a l r o l e o f l a c c a s e i n c u t i c l e s c l e r o t i z a t i o n w i l l , no doubt, r e c e i v e f u r t h e r a t t e n t i o n i n the f u t u r e because i t may be the p r i m a r y p h e n o l o x i d a s e t h a t c o n v e r t s c a t e c h o l a m i n e s i n t o c r o s s - l i n k i n g agents. I n h i b i t i o n o f i n s e c t l a c c a s e s was f i r s t s t u d i e d by Yamazaki (12.24), who found t h a t t h i s enzyme i s i n h i b i t e d by c y a n i d e and d i e t h y l d i t h i o c a r b a m a t e , but i s f a i r l y i n s e n s i t i v e t o t h i o u r e a and c a r b o n monoxide. S t u d i e s by Andersen (8) and B a r r e t t (23) have shown t h a t l a c c a s e s from many i n s e c t s p e c i e s are l e s s s e n s i t i v e t o phenylthiourea, but more s e n s i t i v e to a z i d e t h a n are t y r o s i n a s e s . I n our l a b o r a t o r y , we have a l s o found t h a t l a c c a s e from M. sexta i s l e s s s e n s i t i v e t o p h e n y l t h i o u r e a than i s t y r o s i n a s e , but carbon monoxide has not proven u s e f u l f o r d i s t i n g u i s h i n g the two types o f phenoloxidases (18). Laccase i s r e p o r t e d l y i n h i b i t e d by mono-



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AL


93

p h e n o l s , a l t h o u g h i t can o x i d i z e monophenols t o f r e e r a d i c a l s a t an appreciable r a t e i f o t h e r compounds are p r e s e n t t h a t w i l l r e a c t w i t h the r a d i c a l s and p r e v e n t i n a c t i v a t i o n o f the enzyme ( 2 5 ) . The ability of laccase from M. sexta t o o x i d i z e monophenols i s demon s t r a t e d by i t s o x i d a t i o n o f s y r i n g a l d a z i n e , a compound w i t h two monophenolic groups (18). O n e - e l e c t r o n o x i d a t i o n o f each monophen o l i c group r e s u l t s i n r a p i d rearrangement t o a r e l a t i v e l y s t a b l e quinone methide w h i c h has a pink c o l o r . T o p i c a l a p p l i c a t i o n of syringaldazine to the i n t e r i o r scraped surface o f M. sexta pharate pupal forewing c u t i c l e revealed l a c c a s e a c t i v i t y in situ (18). A l s o , N - a c e t y l - 3 - m e t h o x y - 4 - h y d r o x y p h e n y l e t h y l a m i n e does not i n h i b i t the p a r t i a l l y p u r i f i e d l a c c a s e from M. sexta, but i n stead i s s l o w l y o x i d i z e d by the enzyme (Morgan et. al., unpub l i s h e d data). I t appears l i k e l y t h a t l a c c a s e s are c r i t i c a l enzymes involved i n s c l e r o t i z a t i o n of i n s e c t c u t i c l e . Because o f t h e i r i m p o r t a n c e , a d d i t i o n a l s t u d i e s are needed t o i d e n t i f y e f f e c t i v e inhibitors of laccase t h a t can t h e n be tested for potential insecticidal effects. Peroxidase. P e r o x i d a s e (EC 1.11.1.7, p h e n o l : hydrogen p e r o x i d e o x i d o r e d u c t a s e ) i s i n v o l v e d i n the o x i d a t i o n o f t y r o s y l r e s i d u e s o f proteins f o r the p r o d u c t i o n o f d i t y r o s i n e or t r i t y r o s i n e c r o s s - l i n k s in the e n d o c h o r i o n o f eggs o f Drosophila melanogaster and o t h e r species. T y r o s y l c r o s s - l i n k s are a l s o p r e s e n t i n the highly elastic, rubber-like protein, r e s i l i n , w h i c h i s found i n some f l e x i b l e , a r t i c u l a t i n g regions o f i n s e c t c u t i c l e (26,22). B i t y r o s i n e has a l s o been i s o l a t e d from l a r v a l c u t i c l e o f D. melano gaster and other dipterans and from l a r v a l c u t i c l e o f M. sexta, but not from the i n f l e x i b l e p u p a l c u t i c l e o f M. sexta or f l y puparial cuticle (10). B i t y r o s i n e has a lower o x i d a t i o n p o t e n t i a l than t y r o s i n e . A f t e r one-electron oxidations of both phenolic groups, b i t y r o s i n e r a p i d l y r e a r r a n g e s t o an e l e c t r o p h i l i c q u i n o n o i d s t r u c t u r e t h a t can r e a c t w i t h n u c l e o p h i l e s i n the c u t i c l e . There i s some e v i d e n c e t h a t l a r v a l c u t i c l e s o f d i p t e r a n s and l e p i d o p t e r a n s have p e r o x i d a s e s (9,10), b u t f u r t h e r work i s needed t o v e r i f y those observations. ISOMERIZATION OF QUINONOID COMPOUNDS In a d d i t i o n to phenoloxidases there are o t h e r enzymes i n i n s e c t c u t i c l e w h i c h may help t o c o n t r o l the f o r m a t i o n o f i n t e r m e d i a t e s t h a t are i n v o l v e d i n s c l e r o t i z a t i o n and m e l a n i z a t i o n . These enzymes i n c l u d e a quinone isomerase t h a t c a t a l y s e s the c o n v e r s i o n o f o-qui none t o p-quinone methide and a quinone methide isomerase t h a t catalyses conversion o f p-quinone methide t o α,/3-dehydrocatecholamine. The p-quinone methides o f N-0-alanyldopamine and N-acet y l d o p a m i n e have been proposed as r e a c t i v e i n t e r m e d i a t e s , but they have not been d i r e c t l y o b s e r v e d e i t h e r in vivo or in vitro. However, i t i s r e l e v a n t t o summarize the i n d i r e c t e v i d e n c e f o r t h e i r existence, s i n c e the quinone methides are proposed p r o d u c t s and sub s t r a t e s o f i n s e c t c u t i c u l a r enzymes. A l l e v i d e n c e i s based on the recovery o f c a t e c h o l a m i n e s o r o t h e r d i p h e n o l s t h a t have a c o v a l e n t



94


m o d i f i c a t i o n o f the b e n z y l carbon. Whenever N-acetyldopamine o r N-0-alanyldopamine is oxidized by c u t i c u l a r preparations in vitro, 0-hydroxylation occurs (28-32). The 0-hydroxylation p r o d u c t i s r a c e m i c , s u g g e s t i n g t h a t the h y d r o x y l a t i o n s t e p may be nonenzymatic as would o c c u r w i t h a p-quinone methide o r w i t h a free radical (28 ,.33). The e l e c t r o p h i l i c i n t e r m e d i a t e w i l l a l s o r e a c t w i t h n u c l e o p h i l e s o t h e r t h a n w a t e r , such as methanol o r kynure n i n e , as has been demonstrated i n s e v e r a l l a b o r a t o r i e s (1,31,32,34, 35, Morgan et. al., u n p u b l i s h e d d a t a ) . I f the £-hydroxyl a t e d compound i s r e o x i d i z e d and a quinone methide forms, isomeri z a t i o n c a n be e x p e c t e d t o o c c u r w i t h the h y d r o x y l f u n c t i o n b e i n g converted to a keto function. T h i s has been o b s e r v e d w i t h some model compounds (29., 32,36) . There has so f a r been no s p e c t r a l o r c h r o m a t o g r a p h i c e v i d e n c e f o r the quinone methides o f N - a c e t y l d o p amine and N-0-alanyldopamine, a l t h o u g h the o-quinones o f t h e s e c a t e c h o l a m i n e s can be i s o l a t e d by l i q u i d chromatography (Morgan et. al., unpublished observations). Because o f the l a c k o f d i r e c t e v i d e n c e f o r the quinone methide and i t s f a c i l e r e a c t i v i t y w i t h weak n u c l e o p h i l e s , i t i s assumed t h a t t h i s i n t e r m e d i a t e i s extremely short l i v e d . The mechanism o f d i p h e n o l o x i d a t i o n may also include free r a d i c a l f o r m a t i o n , e i t h e r i n the enzymatic r e a c t i o n i t s e l f o r i n subsequent spontaneous d i s p r o p o r t i o n a t i o n s o r c l e a v a g e s (37., 38). The i n f l u e n c e o f d i p h e n o l i c r a d i c a l s on β c a r b o n r e a c t i v i t y has not been w e l l d e l i n e a t e d . The m e t a s t a b l e r a d i c a l s a r e n o t e a s i l y s t u d i e d i n s o l u t i o n o r wet s o l i d s such as c u t i c l e because t h e y a r e p r e s e n t a t v e r y low c o n c e n t r a t i o n s and undergo r a p i d nonenzymatic reactions. Quinonoid isomerases. ο-Quinone: p-quinone methide isomerases have been found i n c u t i c l e s from f o u r i n s e c t s p e c i e s (36.39.40.41. Table I ) . A quinone tautomerase was a l s o d e t e c t e d i n Sarcophaga bullata l a r v a l hemolymph ( 4 2 ) . A l t h o u g h Sugumaran (4) o r i g i n a l l y p r o p o s e d t h a t the quinone methides a r e produced d i r e c t l y by o x i d a t i o n o f the a l k y l s u b s t i t u t e d c a t e c h o l s , l a t e r s t u d i e s (39.41.43) have shown t h a t a s t r o n g n u c l e o p h i l e ( N - a c e t y l c y s t e i n e ) does n o t r e a c t w i t h the β c a r b o n , b u t i n s t e a d forms an adduct w i t h one o f the r i n g c a r b o n s . T h e r e f o r e , consumption o f the quinone by a s t r o n g nucleophile was proposed t o p r e v e n t f o r m a t i o n o f the quinone methide. A model s u b s t r a t e , 3,4-dihydroxymandelic a c i d , was a l s o o r i g i n a l l y b e l i e v e d t o be o x i d i z e d d i r e c t l y t o p-quinone methide (44), b u t d a t a from experiments u s i n g s h o r t time r e s o l u t i o n s p e c t r o s copy, k i n e t i c s , r a d i o l y s i s , c y c l i c v o l t a m e t r y , chronoamperometry and p r o d u c t a n a l y s i s a r e i n c o n s i s t e n t w i t h the f o r m a t i o n o f a p - q u i none methide d i r e c t l y from an ο-diphenol (45-47). A l l o f t h e s e r e s u l t s i n d i c a t e t h a t the quinone i s the i n i t i a l o x i d a t i o n p r o d u c t . Although quinone methides o f N-acetyldopamine and N - 0 - a l a n y l dopamine have been r e f e r r e d t o as tautomers o f the o-quinones i n the literature, t h e i r spontaneous i s o m e r i z a t i o n r a t e s a r e o f t e n n o t r e p o r t e d o r assumed t o be v e r y low. However, the spontaneous forma tion of Ν-β-alanylnorepinephrine from Ν-β-alanyldopamine-oquinone i s s u b s t a n t i a l , w i t h about a 14% y i e l d i n 10 min i n pH 6 phosphate b u f f e r (34, Morgan et. al., u n p u b l i s h e d d a t a ) .



7.

KRAMER ET AL.


95

A n o t h e r p o s s i b l e type o f i s o m e r i z a t i o n i n v o l v e s c o n v e r s i o n o f the p-quinone methide to an α,^-dehydrocatecholamine. The l a t t e r compound has been i d e n t i f i e d as a m e t a b o l i t e o f N - a c e t y l dopamine during incubation with c u t i c u l a r preparations and an exogenous n u c l e o p h i l e such as an amino a c i d ( 4 8 ) . Andersen and Roepstorff (48) suggested t h a t the n u c l e o p h i l e competes f o r a r e a c tive intermediate i n v o l v e d i n the f o r m a t i o n o f c a t e c h o l a m i n e dimers and t h a t t h i s c o m p e t i t i o n a l l o w s s i g n i f i c a n t amounts o f u n s a t u r a t e d c a t e c h o l a m i n e t o accumulate. Measurable q u a n t i t i e s o f a u n s a t u r a t e d c a t e c h o l a m i n e are not u s u a l l y p r e s e n t i n c u t i c l e . I t may not accumu l a t e because the Κ value for t h i s substrate with c u t i c u l a r lac case i s 60 times lower t h a n t h a t f o r N-acetyldopamine (48)· Al though Sugumaran et. al. (49,50) a l s o demonstrated t h a t r a p i d consumption o f an u n s a t u r a t e d c a t e c h o l a m i n e can o c c u r , Sugumaran (51) p r o p o s e d t h a t t h i s compound i s never formed i n c u t i c l e based on r e s u l t s o f r a d i o a c t i v e t r a p p i n g e x p e r i m e n t s . R e c e n t l y , an enzyme that apparently converts the p-quinone methide t o the α,β-deh y d r o isomer has been d e t e c t e d i n c u t i c u l a r e x t r a c t s from one species (52, T a b l e I ) . T h i s r e s u l t suggests t h a t a d i r e c t c a t e c h o l amine α,/3-desaturation s t e p does not o c c u r , at l e a s t i n puparia o f S. bullata. Andersen (8) p r e v i o u s l y s u g g e s t e d t h a t a d i r e c t α, β-desaturâtion s t e p may o c c u r i n Locus ta migratoria, but a d e s a t u r a t i n g enzyme has not y e t been i d e n t i f i e d . The α,β-deh y d r o c a t e c h o l a m i n e appears t o be an i m p o r t a n t m e t a b o l i t e and i t s r o l e i n s c l e r o t i z a t i o n s h o u l d r e c e i v e more study. The r e l a t i o n s h i p , i f any, between p h e n o l o x i d a s e s and q u i n o n o i d isomerases i s p o o r l y u n d e r s t o o d . U n l i k e the former, the l a t t e r enzymes are not w e l l c h a r a c t e r i z e d i n terms o f c h e m i c a l , p h y s i c a l or k i n e t i c p r o p e r t i e s . The r e a c t i v i t y and s h o r t l i f e t i m e o f s u b s t r a t e s such as p-quinone methides make i t d i f f i c u l t t o e n v i s i o n s i m p l e enzyme i n t e r a c t i o n s w i t h the methides, e s p e c i a l l y when w a t e r and o t h e r n u c l e o p h i l e s are a v a i l a b l e f o r r e a c t i o n . I t may be t h a t the isomerases s i m p l y modulate the a c t i v i t y o f p h e n o l o x i d a s e s so t h a t tautomerization occurs. CONVERSION AND

BLOCKING FACTORS

For many y e a r s the b i o s y n t h e s i s o f m e l a n i n was thought t o r e s u l t from the spontaneous o x i d a t i o n and p o l y m e r i z a t i o n o f dopachrome produced by the t y r o s i n a s e - c a t a l y z e d h y d r o x y l a t i o n o f t y r o s i n e t o dopa and subsequent o x i d a t i o n ( 5 3 ) . In a d d i t i o n to t y r o s i n a s e , however, s e v e r a l enzymatic f a c t o r s have been r e c e n t l y i d e n t i f i e d i n mammalian t i s s u e s t h a t appear to r e g u l a t e melanogenesis a t interme d i a t e s t e p s d i s t a l t o those i n v o l v i n g t y r o s i n e and dopa. The factors include dopachrome conversion factor, dihydroxyindole blocking f a c t o r , dihydroxyindole conversion f a c t o r and dopachrome oxidoreductase (54-59). Dopachrome c o n v e r s i o n f a c t o r c a t a l y z e s the d e c o l o r i z a t i o n o f dopachrome. The mechanism o f t h i s c o n v e r s i o n a p p a r e n t l y i n v o l v e s an i s o m e r i c rearrangement o f a hydrogen atom from one p o s i t i o n o f the dopachrome m o l e c u l e t o a n o t h e r , an i n t r a m o l e c u l a r o x i d o r e d u c t i o n which r e s u l t s i n a tautomeric s h i f t forming 5,6-dihydroxyindole-2-



Sarcophaga bullata

melanogaster

42

Quinone tautomerase, phenoloxidase ΝΑΝΕ

NADA quinone

Hemolymph

21

Larval c u t i c l e extract

Mushroom tyrosinase, quinone isomerase

NADA

Caffeiylmethylamide, dihydroxycaffeiyl me thy1amide quinone, [dihydroxycaffeiyl methylamide quinone methide]

36,12,24

25,26

25,26

41

75

Reference

Dihydroxycaffeiyl methylamide

??

??

Diphenoloxidase, NADA quinone isomerase

Enzyme(s)

Diphenoloxidase, NADA quinone isomerase, NADA quinone methide isomerase

ΝΑΝΕ, NBANE

ΝΑΝΕ, NBANE

ΝΑΝΕ, NADA quinone, insoluble adducts

ΝΑΝΕ

Products

enzymes i n insect tissues.

ΝΑΝΕ, NADA quinone, [NADA quinone methide], a,β-dehydro NADA

NADA, NBAD

NADA, NBAD

NADA

NADA

Substrate

Larval c u t i c l e extract

Larval & c u t i c l e s puparial

puparial c u t i c l e s

Larval &

Calliphora vicina

Diptera

Drosophila

Pharate adult c u t i c l e residue

Periplaneta americana

Dictyoptera

Pupal & adult cuticles

Tissue

Tenebrio molitor

Species

Coleoptera

Order

Table I. Quinonoid isomerizing



Locusta migratoria

Orthoptera

extract

Pharate adult cuticle NADA, NBAD

NADA quinone

NBAD

T r y p s i n i z e d pharate pupal c u t i c l e extract

Colleterial gland

NADA

NADA, NBAD

NADA

N-Acyldopamines

Pharate pupal c u t i c l e residue

Wing c u t i c l e

Larval cuticle

Pupal c u t i c l e , Silk

ΝΑΝΕ, NBANE

ΝΑΝΕ

Laccase

NBANE, [NBANE quinone], [NBAD quinone methide]

Phenoloxidase, ??

??

30,39,41

25,76

40

32

75,76

32

Morgan et. a l . , unpublished data

Diphenoloxidase, NADA quinone: quinone methide isomerase

??

ΝΑΝΕ, NBANE

ΝΑΝΕ,NADA quinone, [NADA quinone methide], insoluble adducts

Diphenoloxidase, laccase, quinone isomerase

ΝΑΝΕ, NADA quinone [NADA quinone methide]

β-Hydroxylated N-acyldopamines

*Bracket i n d i c a t e s only i n d i r e c t evidence a v a i l a b l e f o r formation o f compound. Abbreviations are NADA, N-acetyldopamine; ΝΑΝΕ, N-acetylnorepinephrine; NBAD, N-3-alanyldopamine; NBANE, N-3-alanylnorepinephrine.

Tenodera sinensis

Manduca sexta

Hyalophora cecropia

Dictyoploca japonica

Mantodea

Lepidoptera


^4


98


carboxylic acid (60). T h e r e f o r e , a more c o r r e c t nomenclature f o r dopachrome c o n v e r s i o n f a c t o r i s dopachrome isomerase o r tautomerase. D i h y d r o x y i n d o l e b l o c k i n g f a c t o r b l o c k s the i n d o l i z a t i o n o f quinone imine d e r i v a t i v e s . D i h y d r o x y i n d o l e c o n v e r s i o n f a c t o r c a t a l y z e s the d e h y d r o g e n a t i o n o f 5 , 6 - d i h y d r o x y i n d o l e t o i n d o l e - 5 , 6 - q u i none. Dopachrome o x i d o r e d u c t a s e c o n v e r t s dopachrome t o 5,6-dihy droxy i n d o l e and a l s o may b l o c k 5 , 6 - d i h y d r o x y i n d o l e o x i d a t i o n and subsequent melanogenic reactions. Relatively l i t t l e information i s a v a i l a b l e about the p h y s i c a l , c h e m i c a l and k i n e t i c p r o p e r t i e s o f these proteinaceous factors in mammals. C o n t r o v e r s y about m e l a n i n - r e l a t e d r e g u l a t o r y f a c t o r s has f o c u s e d on whether a c t i v i t y i s due t o unique i n d i v i d u a l p r o t e i n s o r i s o n l y an e x p r e s s i o n o f activities o f a m u l t i c a t a l y t i c enzyme (61,62). For example, d i h y d r o x y i n d o l e c o n v e r s i o n a c t i v i t y i n mice melanoma i s a p p a r e n t l y due t o t y r o s i n a s e , n o t a unique f a c t o r ( 5 6 ) . A l t h o u g h the c o n v e r s i o n o r b l o c k i n g f a c t o r s f o r dopa metabolism are i n c o m p l e t e l y understood i n mammals, even l e s s i s known about these factors i n insects (Table I I ) . Manduca sexta pharate p u p a l integument c o n t a i n s a t y r o s i n a s e t h a t e x h i b i t s d i h y d r o x y i n d o l e c o n v e r s i o n a c t i v i t y and c a t a l y z e s o x i d a t i o n o f d i h y d r o x y i n d o l e t o indole-5,6-quinone (11,63.). The same t i s s u e p o s s e s s e s a dopa quinone imine c o n v e r s i o n f a c t o r t h a t a c c e l e r a t e s the d e c a r b o x y l a t i o n o f dopa quinone imine t o d i h y d r o x y i n d o l e . A s i m i l a r enzyme t h a t d e c o l a r i z e s dopachrome, presumably dopa quinone imine c o n v e r s i o n f a c t o r , has been p a r t i a l l y p u r i f i e d from hemolymph o f Hyalophora cecropia d i a p a u s i n g pupae (64)· I n Bombyx mori, dopa quinone imine c o n v e r s i o n f a c t o r s i n integument and hemolymph o c c u r i n h i g h e s t c o n c e n t r a t i o n s d u r i n g the l a t t e r p a r t o f the f i f t h l a r v a l instar (Aso et. al., unpublished data). I t i s postulated that dopa quinone imine c o n v e r s i o n f a c t o r i n integument p a r t i c i p a t e s i n wound h e a l i n g and/or s c l e r o t i z a t i o n , whereas i n hemolymph i t may f a c i l i t a t e m e l a n i z a t i o n i n the humoral immune system.

Table I I .

C o n v e r s i o n f a c t o r s f o r dopa metabolism

Factor

Species

Dopa quinone imine conversion factor

Dihydroxyindole conversion factor (tyrosinase?)

i n insects

T i s s u e source

Reference

M. sexta

Pharate pupal cuticle

11.63

Β .mori

Hemolymph and cuticle

H.cecropia

Hemolymph o f d i a p a u s i n g pupae

64

M.sexta

Pharate pupal cuticle

11

Aso et al., unpubl. data


7.

KRAMER ET AL.


99

To summarize, dopa quinone imine c o n v e r s i o n f a c t o r s have been d e t e c t e d i n c u t i c l e and/or hemolymph from o n l y t h r e e s p e c i e s o f Lepidoptera. Other k i n d s o f r e g u l a t o r y f a c t o r s such as d i h y d r o x y i n d o l e b l o c k i n g f a c t o r have n o t been d e t e c t e d i n i n s e c t t i s s u e s . The p r e c i s e p h y s i o l o g i c a l r o l e s p l a y e d by c o n v e r s i o n f a c t o r s t h a t g e n e r a t e i n d o l e s i s unknown. They may be m o d u l a t o r s o f r e a c t i o n s associated primarily with melanization.


CROSS-LINK AND PIGMENT STRUCTURES P r o g r e s s has been made r e c e n t l y i n i d e n t i f y i n g bonds between c a t e cholamine a r o m a t i c and a l i p h a t i c c a r b o n t o p r o t e i n n i t r o g e n i n insect cuticle by s o l i d - s t a t e ^H- C-^% d o u b l e - c r o s s p o l a r i z a t i o n (DCPMAS) NMR, (£5) and r o t a t i o n a l - e c h o , double resonance (REDOR) C and N NMR (66,67, C h r i s t e n s e n e t . a l . , unpub l i s h e d d a t a ) . Tobacco hornworm p u p a l e x u v i a e were l a b e l e d by i n j e c t i o n w i t h a c o m b i n a t i o n o f [ r i n g - N 2 ] h i s t i d i n e and e i t h e r [ r i n g e d dopamine or [β- C]dopamine. The DCPMAS difference spectrum o f [ r i n g - " ^ C g ] - l a b e l e d c u t i c l e r e v e a l s a peak a t 135 ppm, u p f i e l d from t h e major oxygenated r i n g c a r b o n peak a t 144 ppm (Fig. 3, m i d d l e r i g h t spectrum). The same peak was o b s e r v e d i n t h e REDOR d i f f e r e n c e spectrum, b u t i t was o b t a i n e d u s i n g l e s s t h a n h a l f the number o f scans r e q u i r e d f o r t h e DCPMAS spectrum. The presence o f a d i f f e r e n c e s i g n a l a t 135 ppm i s c o n s i s t e n t w i t h t h e f o r m a t i o n o f a c o v a l e n t bond between an a r o m a t i c dopamine c a r b o n and a h i s t i d y l ring nitrogen. DCPMAS-NMR of [£- C]dopamine c u t i c l e was n o t s e n s i t i v e enough t o observe c o v a l e n t bond f o r m a t i o n between t h e β c a r b o n o f dopamine and h i s t i d i n e due t o i n h e r e n t l y slow C-N p o l a r i z a t i o n t r a n s f e r and f a s t s p i n - l o c k r e l a x a t i o n . However, an o r d e r - o f - m a g n i tude improvement i n s e n s i t i v i t y , o b t a i n e d by u s i n g REDOR NMR, f a c i l i t a t e d the observation o f a s i z e a b l e C REDOR d i f f e r e n c e s i g n a l a t 60 ppm, w h i c h i s u p f i e l d from t h e major oxygenated a l i p h a l i c c a r b o n peak a t 75 ppm ( F i g . 2, t o p l e f t spectrum). The d i f f e r e n c e s i g n a l i s c o n s i s t e n t w i t h d i r e c t bonding o f t h e β c a r b o n o f dopamine w i t h a r i n g n i t r o g e n o f h i s t i d i n e . We e s t i m a t e t h a t i n M. sexta p u p a l c u t i c l e a p p r o x i m a t e l y t w o - t h i r d s o f t h e bonds t o h i s t i d i n e n i t r o g e n a r e made through c a t e c h o l a m i n e r i n g c a r b o n s , and about one-third through t h e β c a r b o n ( C h r i s t e n s e n et. al. unpub lished) . The two C-N bonds i d e n t i f i e d by NMR a r e c o n s i s t e n t w i t h an i m i d a z o y l n i t r o g e n a t t a c k i n g e i t h e r a p h e n y l c a r b o n o f an o-quinone i n t e r m e d i a t e o r a β c a r b o n o f a p-quinone methide intermedi ate . S o l i d s t a t e NMR has been used t o demonstrate t h e presence o f m e l a n i n - t y p e pigments i n b e e t l e c u t i c l e s ( 6 8 ) . The n a t u r a l abun dance C-NMR d i f f e r e n c e spectrum o b t a i n e d by s u b t r a c t i n g the spectrum o f powdered e l y t r a removed from w i l d - t y p e r e d f l o u r b e e t l e s , Tribolium castaneum, from t h a t o f powdered e l y t r a from the black mutant strain r e v e a l e d t h a t w i l d - t y p e and black e l y t r a have s i m i l a r l e v e l s o f p r o t e i n , c h i t i n and l i p i d , b u t t h a t the black elytra have more melanin or other polyphenolic materials. I t was e s t i m a t e d t h a t a p p r o x i m a t e l y 5% o f t h e t o t a l 1 3

1 5

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.

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F i g . 3. S i n g l e (bottom) and double ( m i d d l e ) c r o s s - p o l a r i z a t i o n magic a n g l e s p i n n i n g (DCPMAS) and r o t a t i o n a l - e c h o , double resonance ( t o p , REDOR) C NMR s p e c t r a o f tobacco hornworm p u p a l e x u v i a e double l a b e l e d by i n j e c t i o n o f [β- C ] dopamine o r [ r i n g - Cg]dopamine t o g e t h e r w i t h [ring- N ] The DCPMAS and REDOR s p e c t r a a r e d i f f e r e n c e s p e c t r a t h a t a r i s e o n l y from those C's d i r e c t l y bonded t o N's. 1 5

h i s t i d i n e

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7.

KRAMER ET AL.


101

a r o m a t i c carbons i n black e l y t r a o c c u r as eumelanin o r o t h e r polyphenols. The s p e c t r a o f w i l d - t y p e e l y t r a , on the o t h e r hand, had more c a r b o n s i g n a l s c h a r a c t e r i s t i c o f a l a n i n e ( b o t h methyl ene and c a r b o n y l c a r b o n s ) . A p p a r e n t l y , the m e l a n i n p r e c u r s o r dopa mine i s i n i t i a l l y d i r e c t e d i n t o the eumelanin pathway i n the black s t r a i n because o f a temporary l a c k o f N - a c y l a t i o n w i t h β-alanine. N-£-Alanyldopamine accumulates more r a p i d l y a t the expense o f dopamine i n the w i l d - t y p e s t r a i n such t h a t m e l a n i z a t i o n o c c u r s t o a much l e s s e r degree. The presence o f c e r t a i n low m o l e c u l a r w e i g h t c a t e c h o l a m i n e m e t a b o l i t e s i n tanned c u t i c l e has been used as i n d i r e c t e v i d e n c e f o r covalent modification of the α and β carbons o f catecho lamines. B e n z o d i o x i n - t y p e dimers o f N-acetyldopamine are p r e s e n t i n i n s e c t c u t i c l e , and the d i m e r i c l i n k a g e i n v o l v e s e t h e r bonds between the p h e n o l i c oxygens o f one monomer and the α and β carbons o f the o t h e r monomer ( 8 ) . M i l d a c i d h y d r o l y s i s o f the dimer pro duces e q u a l amounts o f N-acetyldopamine and 3 , 4 - d i h y d r o x y p h e n y l k e t o ethanol. T h i s k e t o c a t e c h o l i s a l s o a major p r o d u c t o f m i l d a c i d h y d r o l y s i s o f many i n s e c t c u t i c l e s . Sugumaran (69) argues t h a t the presence o f 3 , 4 - d i h y d r o x y p h e n y l k e t o e t h a n o l does not p r o v i d e e v i d e n c e f o r α-carbon m o d i f i c a t i o n o f c a t e c h o l a m i n e . Nevertheless, i t i s u s u a l l y a c c e p t e d t h a t k e t o c a t e c h o l s o r N-acetyldopamine dimers are evidence f o r a , 0 - s c l e r o t i z a t i o n r e a c t i o n s ( 8 ) . The presence o f /3-hydroxylated catecholamines such as N-0-alanylnorepinephr i n e i n the p u p a l c u t i c l e o f M. sexta provides evidence f o r quinone methide or s c l e r o t i z a t i o n (30). Cold d i l u t e acid extracted substantially more 0 - h y d r o x y l a t e d c a t e c h o l a m i n e than d i d n o n - a c i d i c s o l v e n t s , a r e s u l t s u g g e s t i n g t h a t some o f the N-£a l a n y l n o r e p i n e p h r i n e may be a h y d r o l y s i s p r o d u c t o f N - 0 - a l a n y l dopamine which has a r e l a t i v e l y weak c o v a l e n t bond t o the β carbon. Model experiments w i t h manducin, which i s b o t h a hemolymph and c u t i c u l a r p r o t e i n o f M. sexta, have documented the f o r m a t i o n o f c r o s s - l i n k e d m u l t i m e r s o f p r o t e i n f o l l o w i n g o x i d a t i o n o f catecho lamines by p h e n o l o x i d a s e s (Ζΰ,ΖΙ, Thomas et. al., unpublished data). N-0-Alanyldopamine, N-acetyldopamine o r 1,2-dehydro-Nacetyldopamine caused c r o s s - l i n k i n g o f manducin i n the presence o f mushroom t y r o s i n a s e (ZQ,21)· We have a l s o found t h a t these compounds a r e p r e c u r s o r s o f c r o s s - l i n k i n g agents f o r manducin i n the presence of partially purified laccase o r t y r o s i n a s e from M. sexta (Thomas et. al., unpublished data). The c r o s s - l i n k i n g s i t e s o f manducin have n o t been i d e n t i f i e d . S o l i d - s t a t e NMR e v i d e n c e f o r M. sexta cuticle s u p p o r t s bonding o f c a t e c h o l a m i n e a r o m a t i c carbons and a l i p h a t i c carbons (β) t o n i t r o g e n - c o n t a i n i n g s i d e chains of histidine (65,72, C h r i s t e n s e n et. al., unpublished data). A l t h o u g h model experiments and s o l i d - s t a t e NMR s t u d i e s have p r o v i d e d good e v i d e n c e f o r c r o s s - l i n k i n g o f c u t i c u l a r components, n o n c o v a l e n t bonds are a l s o i n v o l v e d i n the s t a b i l i z a t i o n o f the insect exoskeleton. However, the c o n t r i b u t i o n o f these n o n c o v a l e n t i n t e r a c t i o n s t o the p r o c e s s o f s c l e r o t i z a t i o n i s n o t w e l l u n d e r s t o o d

(21).


102



CONCLUDING REMARKS S u b s t a n t i a l p r o g r e s s has been made towards the i d e n t i f i c a t i o n o f c r o s s - l i n k s between polymers i n i n s e c t e x o s k e l e t o n and i n c h a r a c t e r i z a t i o n o f enzymes t h a t a r e r e q u i r e d f o r s y n t h e s i s o f c r o s s - l i n k i n g agents. These agents may be q u i n o n o i d compounds w i t h e l e c t r o p h i l i c carbons i n t h e r i n g and i n t h e α and β p o s i t i o n s o f the s i d e chain. S o l i d - s t a t e NMR has i d e n t i f i e d bonds between the β and r i n g carbons o f c a t e c h o l a m i n e s and the i m i d a z o y l n i t r o g e n o f h i s t i d y l r e s i d u e s o f p r o t e i n . I n s p i t e o f these r e c e n t advances, t h e r e a r e s t i l l many gaps i n our knowledge o f the s u p r a m o l e c u l a r s t r u c t u r e o f i n s e c t c u t i c l e and t h e c h e m i c a l and enzymatic p r o c e s s e s t h a t occur during i t s formation. Acknowledgments T h i s r e s e a r c h was a c o l l a b o r a t i v e i n v e s t i g a t i o n between the U.S. G r a i n Marketing Research Laboratory, A g r i c u l t u r a l Research Service, U.S. Department o f A g r i c u l t u r e , the Department o f Entomology, Kansas A g r i c u l t u r a l Experiment S t a t i o n , Manhattan, KS and the Department o f C h e m i s t r y , Washington U n i v e r s i t y , S t . L o u i s , MO. T h i s i s c o n t r i b u t i o n no. 91-68-A from the Kansas A g r i c u l t u r a l Experiment S t a t i o n , Manhattan, KS 66506. Supported i n p a r t b y N a t i o n a l Science F o u n d a t i o n g r a n t s DCB 86-09717, DIR-8714035, DIR-8720089 and U.S. Department o f A g r i c u l t u r e g r a n t 88-CRCR-3684. We a r e g r a t e f u l t o Drs. J . B a k e r , H. O b e r l a n d e r , R. Howard, S. Andersen and M. B a r r e t t f o r r e v i e w i n g the m a n u s c r i p t . Literature Cited 1. 2.

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