Amino-Acid Sequence, Three Dimensional S t r u c t u r e , Bi o s y n t h e s i s , Rel ease, Physi ologi cal Role,
Receptor Binding A f f i n i t y , and Bi o l o g i cal A c t i v i t y
A K A D E M I S K A V H A N D L I N G
som med v e d e r b ö r l i g t t i l l s t å n d av r ekt or s ä mbe t e t vid Umeå u n i v e r s i t e t f ö r avläggande av medicine doktorsexamen
kommer a t t o f f e n t l i g e n f ö r s v a r a s i Farmakologiska i n s t i t u t i o n e n s f ö r e l ä s n i n g s s a l , onsdagen den 27 maj 1981, kl . 9. 00.
av
S T E F A N 0. E MD I N l eg l äk
Umeå 1981
The A t l a n t i c h a gf i s h, Myxlne. g lu tin o s a , i s t he most p r i mi t i v e v e r t e b r a t e e x t a n t , and i t di ver ged from t he main v e r t e b r a t e e v o l u t i o n a r y chain some 500 mi 11 ion year s ago.
The primary sequence of hagfi sh i n s u l i n shows t h a t i t cont ai ns t he r e s i d u e s implemented f o r e xpr es s i on of a c t i v i t y and t he r e s i d u e s s t a b i l i z i n g t he i n s u l i n monomer and dimer, but not t he hexamer. The primary sequence of hagfi sh p r e p r o i n s u l i n , deduced from the mRNA-cDNA sequence shows l i t t l e homology in sequence of the p r e c u r s o r p a r t s of t he molecule.
However, the sequence cont ai ns t he s t r u c t u r a l requi rements f o r t he t e n t a t i v e f u n c t i o n s , jL .z. v e c t o r i a l di s char ge of the prohormone and a minimum o v e r - a l l s i z e of the p r e c u r s o r . The p r o i n s u l i n convert i ng enzyme(s) seems t o have a s p e c i f i c i t y s i m i l a r t o t h a t of a l l ot he r v e r t e b r a t e s s t u d i e d . The t e r t i a r y s t r u c t u r e of hagf i sh i n s u l i n in t he c r y s t a l i s almost s u p e r imposable on pig i n s u l i n ' s s t r u c t u r e .
The b i o l o g i c a l a c t i v i t y of hagf i sh i n s u l i n is 5% of t h a t of pig i n s u l i n and i t s r e c e p t o r binding a f f i n i t y i s 23% in i s o l a t e d r a t f a t c e l l s . Hagfish i n s u l i n was t he f i r s t p a r t i a l i n s u l i n a n t a g o n i s t on the r a t f a t c e l l i n s u l i n r e c e p t o r . The change(s) in s t r u c t u r e r e s p o n s i b l e f o r the r e duc t i on of a c t i v i t y and binding are not known.
Bi osynt hesi s of hagfi sh i n s u l i n , I n vXJyto, fol lows the p a t t e r n observed in hi gher v e r t e b r a t e s , although a t a much slower r a t e . Unlike the s i t u a t i o n in mammals, hagfi sh i n s u l i n b i o s y n t h e s i s i s not s t i mu l a t e d by gl ucose.
A radioimmunoassay f o r hagf i sh i n s u l i n was developed and the ant i serum c r o s s - r e a c t e d with bovine i n s u l i n t o only 0.01%. The assay was used t o study i n s u l i n r e l e a s e I n vÂJyio. Glucose s t i m u l a t e s i n s u l i n r e l e a s e b u t , u n l i k e the s i t u a t i o n in hi gher v e r t e b r a t e s , amino aci ds do not.
In v iv o , hagfi sh i n s u l i n s t i mu l a t e d the i n c o r p o r a t i o n of ^ C - g l u c o s e and 14C-leuci ne i n t o hagfi sh s k e l e t a l muscle glycogen and p r o t e i n .
The observed s i m i l a r i t i e s , be t we e n hagf i sh and hi gher v e r t e b r a t e s , w i t h regar ds t o i n s u l i n ' s s t r u c t u r e , b i o s y n t h e s i s , r e l e a s e , r e c e p t o r bi ndi ng, and b i o l o g i c a l a c t i v i t y suppor t the concl usi on t h a t , i n s u l i n and i t s proces si ng and e f f e c t o r machi neri es were s t r u c t u r a l l y and b i o l o g i c a l l y well defi ned some 500 mi l l i o n year s ago.
N ew Series N o 66 — ISSN 0346-6612 From the Department of Pathology, University o f Umeå;
the Department of Pathology, University of Lund, Malmö General Hospital, Sweden
Myxine Insulin
Amino-Acid Sequence, Three Dimensional Structure, Biosynthesis, Release, Physiological Role,
Receptor Binding Affinity, and Biological Activity
By
STEFAN O. EMDIN
Umeå 1981
TABLE OF CONTENTS
LIST OF REPORTS, CONSTITUTING THE THESIS 4
INTRODUCTION 5
TAXONOMY 5
EVOLUTION OF THE ENDOCRINE PANCREAS AND INSULIN 6
AIMS OF THE INVESTIGATION 11
MATERIALS AND METHODS
Animals 12
I s o l a t i o n of hagf i sh i n s u l i n 12
Amino-acid sequence a n a l y s i s 12
mRNA i s o l a t i o n and cDNA sequencing 12
C r y s t a l l i z a t i o n and X-ray c r y s t a l l o g r a p h y 13
Pr e pa r a t i on of 125I - i n s u l i n 13
Pr e pa r a t i on of hagf i sh i n s u l i n analogues 13
Bi os ynt he t i c experi ment s 13
S u b c e l l u l a r f r a c t i o n a t i o n 14
Morphology 14
Hagfish i n s u l i n radioimmunoassay 14
I n s u l i n r e l e a s e i n vi&io 14
14 14
Metabolism of C-glucose and C- l euci ne I n vivo 14
RESULTS AND DISCUSSION
I s o l a t i o n o f hagf i sh i n s u l i n 15
Primary s t r u c t u r e of hagf i sh i n s u l i n 15
S t r u c t u r e of p r e p r o i n s u l i n (mRNA) 15
Three-dimensional s t r u c t u r e of hagf i sh i n s u l i n 17
Molecular e v o l u t i o n o f i n s u l i n 17
Degradati on, r e c e p t o r binding a f f i n i t y , and potency of hagf i sh i n s u l i n
in i s o l a t e d r a t f a t c e l l s 19
S t r u c t u r e uc/lòoó a c t i v i t y ; a problem 21
I n s u l i n b i o s y n t h e s i s 22
Conversion and s t o r a g e 23
Radioimmunoassay of hagf i sh i n s u l i n 24
Hagfish i n s u l i n r e l e a s e i n v itn o 24
Ef f e c t s of s t a r v a t i o n 25
Ef f e c t s of hagf i sh i n s u l i n on t he metabolism o f 14C-glucose and
14C- l euci ne in the hagf i sh 25
ACKNOWLEDGEMENTS 26
ABSTRACT 27
REFERENCES 28
REPORTS, CONSTITUTING THE THESIS
This t h e s i s i s based on the fol lowing r e p o r t s , which wi l l be r e f e r r e d t o in t he t e x t by t h e i r Roman numerals:
I. Pe t e r s on, J . D . , S t e i n e r , D. F. , Emdin, S.O. & Falkmer, S.:
The amino acid sequence of the i n s u l i n from a p r i mi t i v e v e r t e b r a t e , the A t l a n t i c hagf i sh (lAyxlnz g lu tin o s a ) . J . Bi ol .Chem.
250:5183-5191, 1975.
I I . Chan, S . J . , Emdin, S. O. , Kwok, S.C.M., Kramer, d.M., Falkmer, S.
& S t e i n e r , D.F . : Messenger RNA sequence and primary s t r u c t u r e of p r e p r o i n s u l i n in a p r i mi t i v e v e r t e b r a t e , t he A t l a n t i c ha gf i s h.
J. Bi ol . Chem. ( i n press ), 1981.
I I I . C u t f i e l d , J . F . , C u t f i e l d , S . M., Dodson, E . J . , Dodson, G.G.,— ^ ---
Emdin, S.O. y& Reynolds, C.D.: S t r u c t u r e and bi o l o g i c a l a c t i v i t y of hagf i s h i n s u l i n . J . M o l . B i o l . 732:85-100, 1979.
IV. Emdin, S.O., Gammel t o f t , S. & Gliemann, J . : Degradat i on, r e c e p t o r bi ndi ng a f f i n i t y , and potency of i n s u l i n from the A t l a n t i c hag
f i s h (My x lm g lu tin o s a ) , determined in i s o l a t e d r a t f a t c e l l s . J . Biol . Chem. 252:602-608, 1977.
V. Emdin, S.O. & Falkmer, S . : Phylogeny of i n s u l i n . Some e v o l u t i o n a ry a s p e c t s of i n s u l i n pr oduct ion with p a r t i c u l a r regard t o t he b i o s y n t h e s i s of i n s u l i n in Myxlne. g lu tin o s a . Acta P a e d i a t r . Scand. , Suppl. 270:15-23, 1977.
VI. Emdin, S.O. & S t e i n e r , D.F. : A s p e c i f i c anti serum a g a i n s t i n s u l i n from t he A t l a n t i c hagf i sh Mi/xxne g lu tin o s a : C h a r a c t e r i z a t i o n of t he ant i ser um, i t s use in a homologous radioimmunoassay, and immunofluorescent microscopy. Gen.Comp.Endocr. 42:251-258, 1980.
VII. Emdin, S.O.: I n s u l i n r e l e a s e in t he A t l a n t i c h a g f i s h , M yxlm g lu tin o s a , I n vXXtio. Gen.Comp.Endocr. ( s u b mi t t e d ) , 1981
VII I . Emdin, S.O.: E f f e c t s of hagf i sh i n s u l i n in t he A t l a n t i c h a g f i s h , My x l m g lu tin o s a . In vivo metabolism of ^ C - g l u c o s e and ^ C - l e u - c i n e , and s t u d i e s on s t a r v a t i o n and glucose loadi ng. Gen.Comp.
Endocr. ( s u b mi t t e d ) , 1981.
x) Due t o a mi s p r i n t in the publi shed v e r s i on, t he middle i n i t i a l has become an "F" i n s t e a d of an "0".
INTRODUCTION
As des cr i bed in d e t a i l in two t hes es ( 1, 2) and a review (3) about 5 ye a r s ago, the i s l e t organ of the A t l a n t i c h a g f i s h , MyxT m glutTnoóa, has a t t r a c ted i n t e r e s t from comparative anat omis ts ever s i n c e the 19th cent ur y. This i n t e r e s t i ncr eas ed when t he hagf i sh p a n c r e a t i c i s l e t c e l l s were analyzed with immunohistochemical and u l t r a s t r u c t u r a l t echni ques and when the obs e r vat i ons made were eval ua t ed a g a i n s t t he background of the evol u t i o n of the g a s t r o - e n t e r o - p a n c r e a t i c endocri ne system ( 1 - 6 ) . The hagf i sh i s l e t organ was found t o occupy a key p o s i t i o n in the phylogeny of the endocri ne pa nc r e as s i nce i t was t he f i r s t s e p a r a t e i s l e t parenchyma in e v o l u t i o n , budding out from t he b i l e duct mucosa and appearing befor e the development of any compact exocri ne pancreas (1- 6) .
When t h i s st udy was i n i t i a t e d , i t was soon r e a l i z e d t h a t al s o the i n s u l i n produced by the hagf i sh i s l e t parenchyma di s pl ayed sever al unusual f e a t u r e s (7). Today, Myxine i n s u l i n i s one of the b e s t known i n s u l i n s , with regard t o t he combined dat a a v a i l a b l e on i t s amino-acid sequence and thr ee- di men
si onal s t r u c t u r e , i t s b i o s y n t h e s i s , r e l eas e, r e c e p t o r bi ndi ng a f f i n i t y , bi o l o g i c a l a c t i v i t y , and phys i ol ogi cal r ol e (8) . An account of
the above p r o p e r t i e s of Myxine i n s u l i n i s the main purpose of t h i s t h e s i s . St udies on speci es v a r i a n t and chemi call y modified i n s u l i n s c o n s t i t u t e the basi s f o r the s t r u c t u r e - f u n c t i o n ' r e l a t i o n s h i p s t h a t seem t o- be import ant
f o r at t empt s to e l u c i d a t e t he hormone's a b i l i t y t o express a c t i v i t y ( 9 , 1 0 ) .
TAXONOMY
The Lat i n name of t he A t l a n t i c hagf i sh was given in 1758 by Carl von Linné ( 1) . MyxTno. g lu tin o ó a belongs t o the phylum CHORDATA, t he subphylum VERTEBRATA9 the c l a s s CVCL0ST0M (or AGMATHA) ( j a w- l e s s f i s h ) , and the order MVXJNOJVEA ( h a g f i s h e s ) (11). As p r e vi ous l y d es cr i bed in d e t a i l ( 1 ) , t he hagf i sh may be c l os e t o a h ypot het i cal common Cambrian or Precambrian a n c e s t o r of a l l v e r t e b r a t e s , t h e r e a f t e r forming an e v ol ut i on l i n e , s e p a r a t e not only from a l l t he ot her more r e c e n t gnasthostomian v e r t e b r a t e s but a l s o from t he second e x t a n t order of t he cyclost omes, v i z . t he Petromyzontia ( t he lampreys). Foss i l remnants i n d i c a t e t h a t t he e v o l u t i o n a r y o r i g i n of t he e x t a n t lampreys i s among the l a t e Ordivi ci an C2.phalaApTd0m0h.phLy l i v i n g
about 500 mi l l i o n y ear s ago, whereas t h a t of t he r e c e n t hagf i s hes - t he PtQAaApidomo/Lpki - l i e s even f u r t h e r back (12). Although i t i s s t i l l a ma t t e r of cont r over s y whether or not t h e cyclostomes r e p r e s e n t r eal ances
t o r s t o the s u p e r c l a s s of gnathostomian v e r t e b r a t e s ( 1 , 1 2 ) , MyxÀne. g lu tin o s a i s , " i n f a c t , one of t he few s ur v i v i n g r e p r e s e n t a t i ves of t h a t v e r t e b r a t e group which f i r s t appears in t he geologi cal d e pos i t s " (12) , and thus o f co n s i d e r a b l e phyl ogenet ic and e v o l u t i o n a r y i n t e r e s t ( 1 - 6 ) . I t
should be poi nt ed o ut , a l r eady a t t h i s j u n c t u r e , t h a t s t r u c t u r a l and func
t i o n a l f e a t u r e s found in t he hagf i s h cannot à p r i o r i be assumed t o r e p r e s e n t c o ndi t i ons once p r e s e n t in the ostracoderms and even e a r l i e r in e v o l u t i o n , si n c e e x t a n t Myxinoids have had as much time t o evolve and adapt as the e n t i r e group of Gnathostomes has taken f o r i t s p h y l e t i c d i f f e r e n t i a t i o n (13) .
EVOLUTION OF THE ENDOCRINE PANCREAS AND INSULIN
Despit e t he f a c t t h a t t he . i s l e t organ (endocr i ne pancreas) did not appear in e v o l u t i o n u n t i l t he v e r t e b r a t e s evolved, t h e r e i s growing e vi de n
ce t h a t i n s u l i n could have been p r e s e n t long befor e ( 8 , 1 4 ) . The evidence i s l a r g e l y based on radioimmunoassays and immunocytochemistry, using ant i ser a
a g a i n s t mammalian i n s u l i n s (14). There a r e no publ i shed dat a on t he chemical s t r u c t u r e of t he presumed p r e v e r t e b r a t e i n s u l i n s , and the s t r u c t u r e of such i n s u l i n s may, indeed, be highly d i f f e r e n t from v e r t e b r a t e i n s u l i n s , e s p e c i a l l y s i n c e c e r t a i n growth f a c t o r s l i k e IGF I and II ( i n s u l i n l i k e growth f a c t o r ) , r e l a x i n and p o s s i b l y NGF (nerve growth f a c t o r ) and MSA (multi pi i e at i ng s t i m u l a t i n g a c t i v i t y ) (15,16) show c o n s i d e r a b l e s t r u c t u r a l s i m i l a r i t i e s wi t h i n s u l i n . I t has been suggested t h a t the members of t h i s i n s u l i n family ar e uni t ed by a common a n c e s t o r (17). In a d d i t i o n , t h e r e i s evidence of an e v o l u t i o n a r y r e l a t i o n s h i p between i n s u l i n and t he s e r i n e pr ot e a s e s (18).
S t e i ne r (19) has s pe c ul a t e d t h a t l i mi t e d enzymatic d i g e s t i o n of t he c e l l s u r f a c e s could have been the most p r i mi t i ve c e l l - c e l l communication. Such ..
a system, o p e r a t i n g through a process analogous t o down- r egul at i on, could have become a dj us t e d t o u t i l i z i n g p r o t e o l y t i c f r agment (s) t h a t i n t e r a c t e d f avour ably with r e g u l a t o r y c e l l s u r f a c e p r o t e i n . Hence, a b as i c r e p e r t o i r e of p r o t e i n s t h a t had communicative a b i l i t i e s could have been generat ed and f u r t h e r d i v e r s i f i e d through gene d u p l i c a t i o n . I f s o , t h e the dual occurrence of many g a s t r o - e n t e r o - p a n c r e a t i c hormones in the gut and the nervous system i s perhaps not so s u r p r i s i n g (19) .
With t he aid of radioimmunoassay, i t has r e c e n t l y been claimed t h a t
" i n s u l i n " i s p r e s e n t a l r eady in u n i c e l l u l a r eukaryot es and p r o k a r y o t e s , alt hough in exceedi ngly low c o n c e n t r a t i o n s (about one molecule per organism or even l e s s in the case of E. c o l i ) ( 20, 21, 22) .
In protostomi an i n v e r t e b r a t e s , i n s u l i n i mmunoreacti vit y has been found in the nervous system of the tobacco hookworm Manduca A cxta ( 23) , and l a t e r i n s u l i n producing c e l l s were de t e c t e d in the nervous system of the blow
f l y , CaLLcpkofia vom ttotiia (24) . The immunoreactive substance was f u r t h e r e x t r a c t e d and p a r t i a l l y p u r i f i e d , and i t was found t o d i s p l a c e ^ ^ I - i n s u l i n from l i v e r plasma membrane r e c e p t o r s and t o have i n s u l i n - l i k e b i o l o g i c a l a c t i v i t y on i s o l a t e d r a t f a t - c e l l s (25) . Recent l y, i n s u l i n producing c e l l s have been found al s o in t he h o v e r f l y , EtuL&taJLu a m z iu (14) . The precence of pept i de hormones p a r t i c u l a r i t y in t he c e n t r a l nervous system c o r r e l a t e s well with the sugges ti on t h a t al i ment ar y t r a c t pol ypept i de hormones have o r i g i n a t e d from the neural ectoderm as t r a n s m i t t e r s or modul ators and t h a t they l a t e r have accept ed a new hormonal r o l e as the g a s t r o - i n t e s t i n a l t r a c t developed ( 5, 2 6 ) . Havrankova a t a t . (27) r e por t e d an enrichment of i n s u l i n in the r a t b r a i n , and i t was suggested t h a t i n s u l i n might be s ynt hes i zed i n s i d e t he nervous system. La t e r , Eng and Yalow (28) f a i l e d t o confirm t h i s o bs er vat i on and i t remains an open que s t i on i f , in higher v e r t e b r a t e s , t h e r e ar e hi gher c o n c e n t r a t i o n s of i n s u l i n in t he br ai n than can be expect ed when c o r r e c t i o n has been made f o r i n t r a - v a s c u l a r and r e c e p t o r bound i n s u l i n . There i s no fi rm morphological suppor t f o r i n s u l i n c e l l s in the nervous system of mammals.
In c e r t a i n a d u l t protostomi an and deut er ost omi an i n v e r t e b r a t e s , i n s u l i n c e l l s have been found as di s semi nat ed c e l l s of open type in t he mucosa of t he al i ment ar y t r a c t (Fi g. 1) ( 5) . The o t h e r i s l e t hormones appear t o be p r e s e n t in the gut as well (ctf. 14). The gut i n s u l i n c e l l s in a mollusc responded t o glucose loadi ng by d e g r a n u l a t i o n , and i n s u l i n i mmunor eacti vit y was es t i mat ed in t he hemolympn. Moreover, t he t i s s u e glycogen l evel was suggested t o be under i n s u l i n c ont r ol (29). The i s o l a t i o n and p u r i f i c a t i o n of i n v e r t e b r a t e i n s u l i n has been hampered by t he low amount of i n s u l i n p r e s e n t in heterogenous t i s s u e s . , I t i s p o s s i b l e t h a t complementary nucleo
t i d e probes from c e r t a i n sequences of i n s u l i n mRNA could help in i d e n t i fyi ng i n s u l i n - l i k e cDNA sequences ( I I ) .
In v e r t e b r a t e s i t has been observed t h a t t he ev o l u t i o n of t he endocri ne
GUT LUM EN
iiili::; G L U C A G O N / i INSULIN CELL G A S T R IN CELL
! P P -C E L L
I
SO M A TO STA TIN CELLFIG. 1. P r e - p a n c r e a t i c gut (Amphioxus and ot h e r deut er ost omi an i n v e r t e b r a t e s ) . There i s no i s l e t organ. Zymogen and hormone c e l l s are found in t he mucosa.
B ILE D U C T
B U T LUMEIM
y n m i S ï ï i T H i i i i i H i a n i n i
FIG. 2. The g a s t r o - e n t e r o - p a n c r e a t i c endocri ne system of t he hagfish*
MyxÀne. g lu tin o s a . The i s l e t organ has no exocri ne parenchyma and con
t a i n s mostly i n s u l i n c e l l s and few s oma t os t a t i n c e l l s . There a r e no i n s u l i n c e l l s in t he gut mucosa but a few s omat os t at i n c e l l s as well as glucagon and PP c e l l s of open type.
IN S U L IN C EL L
■
S O M A T O S T A T IN C E L L ( O P E N )•
S O M A T O S T A T IN C E L L ( C L O S E D )^ P P - C E L L
G L U C A G O N / G A S T R IN C E L L ( O P E N )
FIG. 3. Composite p i c t u r e of t he i s l e t organ in the A t l a n t i c ha gf i s h, MljxIykl g l u u t i n o A a , showing i t s main gr os s , l i g h t mi croscopi c, and u l t r a - s t r u c t u r a l f e a t u r e s .
a) The i s l e t organ (arrows) appears t o the naked eye as a whi t i s h swel l ing around t he s h o r t common b i l e duct (BD) a t i t s j u n c t i o n with t he gut. G=gut.
GB=gall bl adder .
b) Transver se s e c t i o n of the common b i l e duct (BD) with i t s surrounding s o l i d ne s t s of i s l e t parenchymal c e l l s , obviously budding out from the b i l e duct mucosa. There i s no exocri ne a c i n a r p a n c r e a t i c parenchyma, whatsoever.
This i s l e t organ i s from a small young h a gf i s h; consequentl y ( 2) , i t i s l acki ng those c y s t i c c a v i t i e s t h a t c h a r a c t e r i z e many i s l e t - c e l l lobul es of t he i s l e t organ of the a d u l t hagf i sh (Fi gs. 3c and d ) . x 300 (approx).
c and d) I s l e t l o b u l e s , immunostained by t he PAP ( p e r o x i d a s e - a n t i - p e r o x i dase) procedure (5) t o v i z u a l i z e i n s u l i n (Fig. 3c) and s oma t os t a t i n (Fi g. 3d) producing parenchymal c e l l s , c o n s t i tuf ng about 95 and 5%, r e s p e c t i v e l y (6) . Glucagon and PP c e l l s do not occur in the hagf i sh i s l e t organ (cf_. Fig. 2) . Most of the t a l l columnar c e l l s , surrounding the c y s t i c c a v i t i e s ~ a n d occu- ri ng in the l obul es of the parenchymal c e l l s in the i s l e t organ of a d u l t hagf i sh (1, 2) , are of non-endocrjne t y p e . x 700 (approx),
e and f ) Elect r on micrographs of t he s e c r e t o r y granul es of the i n s u l i n (Fig. 3e) and the s omat os t at i n (Fi g. 3f) c e l l s , r e s p e c t i v e l y . Fig. 3e a l s o gives examples of the s o - c a l l e d c o n n e c t i v e - t i s s u e s e c r e t i o n t h a t has been descr i bed in endocri ne organs of j a w- l e s s f i s h ( c f . 3) , where s e c r e t o r y granul es seem t o fuse with f i b r e s of c o n n e c t i v e - t i s s u e type. The i n s u l i n granul es (Fig. 3e) ar e r a t h e r pleomorphic and e l e c t r o n dense, whereas the s e c r e t o r y granul es of t he somat os t at i n c e l l s (Fi g. 3f) are l e s s e l e c t r o n dense and s p h e r i c a l , e x 23,000 (appr ox. ) f x 9,000 ( appr ox. ) .
pancreas fol lows a st epwi se p a t t e r n , with regard t o t he occurrence of the four i s l e t hormones (5) . In cyclostomesa compact endocri ne pancreas i s found, e i t h e r around t he b i l e duct ( h a g f i s h e s ) , or in t he submucosa of t he gut
(l ampreys). The endocrine pancreas of cyclostomes cont ai ns agr anul ar c e l l s , i n s u l i n c e l l s , and few (about 1-5%) s o ma t os t a t i n c e l l s (Fi g. 2 and 3). At t h i s st age of e v ol ut i on the c e l l s s t o r i n g t he ot her two i s l e t hormones; PR and glucagon, remain in the gut mucosa as c e l l s of open type. In a d d i t i o n , the gut of the hagfi sh appears t o cont ai n s o ma t o s t a t i n , GIP, gastrin/CCK, and neur ot ensin c e l l s as demonstrated by immunological methods (14).
t u r e of hagf i sh i n s u l i n wi l l be d e a l t with l a t e r on in t he t e x t .
The next s t e p in t he ev o l u t i o n of t he endocri ne pancreas appears t o be a t the l evel of c a r t i l a g i n o u s holocephalan f i s h ( 5) . Only a few e x t a n t s p e c i es e x i s t and they have a compact pancreas with both exo- and endocri ne parenchyma (Fig. 4) . The l a r ge i s l e t s are duct a s s o c i a t e d and cont ai n i n s u l i n , somat os t at i n and glucagon c e l l s . The pancreas i s s i t u a t e d c l os e t o the spl een and i s connected t o the gut with a long duc t . There i s no s t r u c t u r a l inf ormat i on a v a i l a b l e on i n s u l i n from hol ocephalan s p e c i e s .
P A N C R E A T I C
I N S U L IN C E L L Q L U C A Q O N C E L L I S O M A T O S T A T I N
P P C E L L
O U T L U M E N
T n n i i i i n a r m n i m T T T
FIG. 4. The g a s t r o - e n t e r o - p a n c r e a t i c endocrine system of t he holocephalan r a t f i s h , Ckùna&ia monò&ioòa. or tìydAolaguA c o t t i c i . An exocr i ne p a n c r e a t i c gland is p r e s e n t , s i t u a t e d cl os e to t he spl een but widely sepa r a t e d from t he gut. A long s l e nde r duct connects t he gland with t he gut. PP c e l l s are p r e s e n t , but are mainly in the gut mucosa with occasi onal c e l l s in the p a n c r e a t i c duct epi t hel i um. Glucagon c e l l s are p r e s e n t in t he endocri ne pancreas which i s now a t h r e e hormone organ. There are no i n s u l i n c e l l s in t he gut mucosa but t h e r e ar e glucagon and s o ma t os t a t i n c e l l s in a d d i t i o n t o t he PP c e l l s .
In elasmobranchian c a r t i l a g i n o u s f i s h a d i s t i n c t stomach and duodenum appear f o r t he f i r s t time in v e r t e b r a t e e v o l u t i o n . The compact pancreas i s c l o s e l y apposed t o the duodenum. The i s l e t parenchyma has now become a f o u r - hormone organ and PP c e l l s are p r e s e n t ( 5) (Fig.5). Apart from compositional dat a on i n s u l i n and glucagon from the spiny d o g f i s h , Squaluò a c a n tfv ù u , t h e r e i s no s t r u c t u r a l inf ormat ion on the elasmobranchian i s l e t hormones.
In t he l a r ge group on bony f i s h a four hormone i s l e t i s found, with c h a r a c t e r i s t i c s of t h a t in mammals. The PP c o n t a i ni ng i s l e t s appear t o be l oc a t e d near t he duodenum and seems t o be absent in t he s p l e n i c Brockman body ( 5) . A s i m i l a r topogr aphi c d i s t r i b u t i o n i s found in mammals i ncl udi ng man (30). Above t he l evel of t he bony f i s h ( i . e . amphibians, r e p t i l e s , b i r d s , and mammals) t h e r e i s no gross change of i s l e t o r g a n i s a t i o n of hormo
nal cont ent s as t he p h y l e t i c d i f f e r e n t i a t i o n proceeds (14). The s t r u c t u r e s of sever al f i s h i n s u l i n s , as well as t he s t r u c t u r e s of sever al r e p t i l i a n , a vi a n, and mammalian i n s u l i n s ar e known (17).
PANCREATC
D U C T H I I N S U L I N C E L L
O U T L U M E N
FIG. 5. The g a s t r o - e n t e r o - p a n c r e a t i c endocri ne system in an elasmobranch c a r t i l a g i n o u s f i s h . The pancreas i s c l o s e l y a s s o c i a t e d with t he gut.
Numerous PP c e l l s appear in the endocrine pancr eas , t o g e t h e r with i n s u l i n , glucagon and s o ma t os t a t i n c e l l s . Except f o r i n s u l i n c e l l s , t he i s l e t hormo
ne c e l l s are a l s o p r e s e n t in t he mucosa of t he i n t e s t i n e .
AIMS OF THE INVESTIGATION
1. To determi ne t he primary and the t e r t i a r y molecular s t r u c t u r e of the i n s u l i n e x t r a c t e d from the i s l e t organ of t he A t l a n t i c h a g f i s h , Myxlne.
gtuùtnoóa.
2. To give some experi ment al as pect s on the b i o s y n t h e s i s of Myxine i n s u l i n . 3. To st udy t he r e c e p t o r binding a f f i n i t y and b i o l o g i c a l a c t i v i t y of Myxine
i n s u l i n in mammalian t e s t systems and t o c o r r e l a t e the r e s u l t s with the i n s u l i n s t r u c t u r e .
4. To develop a homologous r a d i o-immunoassay f o r Myxine i n s u l i n . 5. To st udy the r e l e a s e of Myxine i n s u l i n â j l v â j ü i o.
6. To t r y to get an idea of the physol ogical r o l e of Myxine i n s u l i n in t he hagfi sh by some experi ment al s t u d i e s .
MATERIALS AND METHODS
Animals
Adult hagf i sh were caught a t the Kr i s t i ne be r g Marine Biology S t a t i o n , f o r sever al year s a t a l l seasons in the Gullmar f i o r d on the Swedish West Coast. A d e t a i l e d account of c a t c hi ng, handli ng and keeping of t he animals has been given by Östberg ( 1 ) , and f u r t h e r d e t a i l s are found in the i n d i v i dual papers ( I , I I , V I I , and VI I I ) .
I s o l a t i o n of hagf i sh i n s u l i n
Hagfish i n s u l i n was e x t r a c t e d with a c i d- et hanol and p u r i f i e d by gel f i l t r a t i o n on Bio-Gel P-30 columns, e l u t e d with 3 M a c e t i c acid ( I ) . Fur t her p u r i f i c a t i o n , when neces s ar y, was accomplished by ion-exchanqe chromatoqraphy on OAE-Sephadex A-25 in 0.05 M NH4 Cl pH 8. 60, in 60% ethanol with a l i n e a r . C l ~ - g r a d i e n t ( I V ).
Amino acid sequence a n a l y s i s
Edman degr adat ion of hagfi sh i n s u l i n and C-pepti de was c a r r i e d out manually using a semi-micro procedure (31 and I ) . Labeled hagfi sh p r e pr oi n- s u l i n was subj e c t e d t o automated Edman degr adat i on in a Beckman 890 C sequencer ( I I ) .
Pr e p r o i n s u l i n mRNA i s o l a t i o n and cDNA sequencing
Hagfish i s l e t RNA was e x t r a c t e d and polyA-enriched mRNA was i s o l a t e d usi ng o l i g o ( d T ) - c e l l u l o s e ( I I ) . DNA sequence a n a l y s i s was performed a c c o r ding t o Maxam and G i l b e r t (32). The methods of c e l l - f r e e t r a n s l a t i o n ,
cloni ng and h y b r i d i z a t i o n are descr ibed in one of t he most r e c e n t r e p o r t s ( I I ) .
C r y s t a l l i s a t i o n and X-ray crys t al l ogr aphy.
Hagfish i n s u l i n was c r y s t a l l i z e d under z i n c - f r e e condi t i ons in e i t h e r c i t r a t e or a c e t a t e buf f er ed aqeous s o l u t i o n s cont ai ni ng acet one, pH 5. 2- 6. 0 (33 and I I I ) . Heavy atom d e r i v a t i v e s were obt ai ned with l e a d , uranyl and gold cyanide ( I I I ) . The methods of dat a c o l l e c t i o n , phase c a l c u l a t i o n and ref inement are given in one of t he r e p o r t s ( I I I ) .
Pr epar at i on of 125I - i n s u l i n
I n s u l i n was i odi na t e d with 125I (The Radiochemical Cent re, Amersham) using chloramine T ( 3 4 , IV, and VI). The assays f o r i n s u l i n r e c e p t o r bi ndi ng, b i o l o g i c a c t i v i t y and degr adat i on have been descr i bed (IV).
Pr epar at i on of hagf i sh i n s u l i n analogues
Des-B^-j-hagfish i n s u l i n was prepared as des cr i bed (IV). Des B3Q-3 1 ” hagf i sh i n s u l i n was prepared from 0. 9 mg d e s - B^ - a n a l o g u e by i ncubat i on with 90 yg carboxypept i dase B (Sigma type I, DFP- t r eat ed) i n 0.1 M di met hyl - a l lyl-amine-HCl pH 7.2 f or 45 min a t 25°C. The d i g e s t was a c i d i f i e d and gel f i l t e r e d over a Bio Gel P-30 column (1 x 40 cm). Amino acid a n a l y s i s showed 2.7 ( pr edict ed;3) lysine r e s i due s in des B ^ - i n s u l i n and 1.97 (pr edi ct ed; 2) l y s i n e r e s i d u e s in the gel f i l t e r e d d i g e s t . The b i o l o g i c a l a c t i v i t y and t he r e c e p t o r bi nding (IV) was measured in i s o l a t e d r a t f a t c e l l s . The
a c t i v i t y was 84-9%. (S.D.; n=5) and the binding was 80% (mean of two e x p e r i ments) of t h a t of n a t i v e hagfi sh i n s u l i n .
B i o s ynt he t i c experiments
In the b i o s y n t h e t i c experiments t he i s l e t s were removed and trimmed from adherent t i s s u e (VII) and f i n a l l y cut i n t o 2-4 pi ece s . All the i s l e t pieces f o r each experi ment were pooled and then di vi ded i n t o d i f f e r e n t tubes and i ncubat ed, with occasi onal a g i t a t i o n . The i ncubat i on medium (VII) was desi gned t o correspond t o hagf i sh plasma (35) . I t contai ned (per ml);
p e n i c i l l i n 100 U, fungizone 0.25 yg and st reptomyci n 100 yg. Before use, the medium was s t e r i l i z e d by f i l t r a t i o n . I n i t i a l l y , bi car bonat e (5mM) was used and t he medium was i n t e r m i t t e n t l y gassed with 97% 02/ 3% C02 . La t e r , b i car bonat e was exchanged f o r N- 2- hydr oxyet hyl pi per azi ne- N- 2- s ul f oni c acid (HEPES), wi t hout observing any adverse e f f e c t s . For the i ncubat i ons 0.1 ml medium with 5 yCi L- ( 4 , 5 - H) l euci ne (The Radiochemical Centr e, Amersham) 3 per i s l e t was used. The remaining unl abel ed amino aci ds were added in c o n c e n t r a t i o n s accordi ng t o Eagle (36). I s l e t s were then e x t r a c t e d f o r
i n s u l i n as descr ibed ( I ) . Bovine i n s u l i n and unlabel ed L- l euc i ne (both 0 . 5 - Img) were i ncl uded in t he e x t r a c t i o n procedure. The i s l e t e x t r a c t s were f r a c t i o n a t e d on 100x1 cm Bio-Gel P 30 columns e l u t e d with 3 M a c e t i c aci d and the r a d i o a c t i v i t y counted. Before gel f i l t r a t i o n some bovine serum albumin and L- l euc ine were added t o t he sample. The p o s i t i o n s of t he void volume and the bovine i n s u l i n peak were determined by readi ng the absorbance a t 276 nm.
S u b c e l l u l a r f r a c t i o n a t i o n
Hagfish i s l e t s were homogenized in a small g l a s s - Te f l o n homogenizer in a bu f f e r con t a i n i n g ; 10 mM KH^PO^S mM NaCl, 1 mM CaCl2 , 0.25 M s uc r os e , 1% (w/v) Fi c o l l , and 0.5% (w/v) bovine serum albumin, pH 6. 0 . The homogenate was c e n t r i f u g e d in a Beckman SW 56 r o t o r as des cr i bed (V), and t he amount of i n s u l i n was es t i mat ed in each f r a c t i o n (VI). The c ont e nt s of the f r a c t i o n s were a l s o judged from e l e c t r o n micrographs. F i n a l l y i s l e t s were puls e- chased with 3H-leucine and processed as above, and t he f r a c t i o n s were then e x t r a c t e d and gel f i l t e r e d ( V ) .
Morphology
Ti ssue f o r t r a ns mi s s i on e l e c t r o n microscopy (V) and immunofluorescence (VI) were processed as d es cr i bed (1 and V).
Hagfish i n s u l i n radioimmunoassay
The development of t he radioimmunoassay and t he r e l a t e d methods ar e d es cr i bed s e p a r a t e l y (VI).
I n s u l i n r e l e a s e I n v W io
Hagfish i s l e t organs were i ncubated e i t h e r s t a t i c a l l y or in a p e r i - f us i on system (VII). Insulir> r e l e a s e was measured with t he i n s u l i n r a d i o immunoassay (VI).
Metabolism o f 14C-glucose and 14C- l euci ne I n vivo
The met abol i c f a t e s of t he two i s ot ope s in the presence of i n s u l i n was st u d i e d In vivo as s p e c i f i c a c t i v i t i e s of l i v e r and s k e l e t a l muscle p r o t e i n or glycogen. The assays of t h e s e along with the assays of t r i g l y c e r i d e s , f r e e f a t t y a c i d s , gl ucos e, and a-ami no- ni tr ogen were made by conventi onal methods and ar e des cr i bed s e p a r a t e l y ( VI I I ) .
RESULTS AND DISCUSSION I s o l a t i o n of hagf i sh i n s u l i n :
Hagfish i s l e t organs were r i ch in i n s u l i n and a f t e r e x t r a c t i o n and gel f i l t r a t i o n c o n s i s t e n t y i e l d s of 1 mg i n s u l i n per g t i s s u e (wet weight) were obt ai ned. The simple procedure followed ( I ) gave i n s u l i n p r e p a r a t i o n s 80-90%
pure, contaminated with two bands, more a c i d i c than hagf i s h i n s u l i n on p o l y acryl ami de gel e l e c t r o p h o r e s i s (37) a t pH 8. 7. Af t er e l u t i o n from t he gel s they showed i n s u l i n immunoreacti vit y and amino aci d compositions i n d i s t i n g u i s h a b l e from hagfi sh i n s u l i n and t hey were presumed t o r e p r e s e n t i n s u l i n mono- and didesamidoforms. Ion exchange chromatography (IV) gave a pure i n s u l i n , a s judged from gel e l e c t r o p h o r e s i s a t aci d and ba s i c pH. When using endogenously l abel ed H-hagfish i n s u l i n t he recovery o f t he gel f i l t 3 r a t i o n s t e p was 69.9 - 3.1% (S.D.;n=4) and 88-94% of l abel ed i n s u l i n was recovered in the f i na l p r e c i p i t a t e when c a r r i e d through the e x t r a c t i o n procedure in the presence of un t r e a t e d whole i s l e t s . Attempts t o p u r i f y hagf i sh p r o i n s u l i n were hampered by the low amounts (1-2% of i n s u l i n , as judged by i mmunoreactivit y) p r e s e n t in i s l e t s , and c ons i der abl e l os ses duri ng p u r i f i c a t i o n .
Primary s t r u c t u r e of hagf i sh i n s u l i n
Hagfish i n s u l i n d i f f e r s in 17 out of 51 r e s i d u e s and i s one of the most hi ghl y s u b s i t u t e d n a t u r a l i n s u l i n s (I and I I ) . On t he ot h e r hand, 23 of t he 24 r e s i d u e s known t o be i n v a r i a n t among ot h e r i n s u l i n s are preserved ( I ) . These r e s i due s incl ude t he dimer forming r es i dues ( 38) , t he p u t a t i v e r e c e p t o r s i t e (9) and the regi on suggested r e s p o n s i b l e f or negat i ve co
op e r a t i v i t y (39). Among t he r es i dues r e s p o n s i b l e f o r Zn- i ns ul i n hexamer formati on a number of s u b s t i t u t i o n s have occurred in hagf i sh i n s u l i n , i n cl udi ng the z i n c - c o o r d i n a t i n g B-jq h i s t i d i n e r e s i d u e . Hagfish i n s u l i n shows f e a t u r e s common t o both mammalian and f i s h i n s u l i n s but does not seem t o be s p e c i f i c a l l y r e l a t e d t o e i t h e r of the groups ( I ) . This i s c o n s i s t e n t with t he cyclostomes di ver ging from the gnathostomes befor e the d i v e r s i on of mammals and f i s h . By using t he c a l c u l a t e d r a t e of evol ut i on f o r i n s u l i n (40) i t i s p o s s i b l e to c a l c u l a t e (50) t h a t the cyclostomes and the gnatho
stomes (50) s e pa r a t e d some 540 mi l l i o n y ear s ago.
S t r u c t u r e of preproinsulin(mRNA)
The mRNA of hagf i sh p r e p r o i n s u l i n i s over 900 n u c l e o t i d e s long, due t o
t he presence of a l ar ge (500 n u c l e o t i d e s ) 3#n o n - t r a n s l a t e d regi on. The nu c l e o t i d e sequence homology, in comparison with human, r a t I and I I , c h i c ken and a n g l e r f i s h p r e p r o i n s u l i n mRNAs, ar e r e s p e c t i v e l y ; pr epept i de 25%, B-chain 48%, C-peptide 19%, and A-chain 57% ( I I ) . This c l e a r l y confirms t h a t t he r a t e s of evol ut i on can d i f f e r markedly between regi ons with d i f f e r e n t f unc t i ons in a s i n g l e p r o t e i n . The N-terminal pr epept i de e x h i b i t s f e a t u r e s t h a t have been consi dered import ant f o r v e c t o r i a l di s char ge through the rough endoplasmic ret i cul um (41). The p r epept i de cont ai ns a hydrophobic c e n t e r , and a t ur n near the B-chain N-terminus can be p r e d i c t e d (42). The C-pepti de i s s i m i l a r in lengt h and general composition, and seems t o r e t a i n many of the non- pol ar r e s i d u e s in the N-terminal h a l f of t he molecule, but has no s i g n i f i c a n t homology with t he C- pepti de of hi gher v e r t e b r a t e s . I t i s p o s s i b l e t h a t the C-pepti de, in a d d i t i on t o i t s f unct i on in providing c o r r e c t o r i e n t a t i o n f or s u l fhydryl o x i d a t i o n , a l s o ser ves as a spacer in keeping a minimum over al l lengt h of t he p r o i n s u l i n mol ecule. The double b a s i c r e s i due s l i nki ng t he C-pepti de with t he A and B-chains in the pro
i n s u l i n moiety e x i s t e d al r e a d y in h a g f i s h . Hence, t he s p e c i f i c i t y of t he converting enzyme system has remained un a l t e r e d t hr oughout v e r t e b r a t e e v o l ut i on.
■
Fig. 6. Hagfish i n s u l i n c r y s t a l s ( s l i g h t l y l e s s than 1 mm), grown in a cet o- n e - c i t r ä t e bu f f e r a t pH 6. 0 in t he absence of zi nc.
Three-dimensional s t r u c t u r e of hagf i sh i n s u l i n
Hagfish i n s u l i n forms t e t r a g o n a l c r y s t a l s (Fi g. 6) with one molecule per asymmetric u n i t and i s organi zed as a p e r f e c t l y symmetrical dimer (i n c o n t r a s t t o 2Zn and 4Zn pig i n s u l i n s ) , l yi ng on a t w o - f o l d - c r y s t a l l o - gr aphic axi s (33, 43). As could be p r e d i c t e d from t he primary sequence, t h e r e i s no i n d i c a t i o n of hi gher or der s t r u c t u r e s . The r e s o l u t i o n of t he dat a r e por t e d here i s 3.1 Â ( I I I ) , but i s being extended t o 1.9 Â spacing (44). Despit e t he d i f f e r e n c e s in sequence and a g g r e g a t i o n , pig and hagf i sh i n s u l i n have c l o s e l y s i m i l a r s t r u c t u r e s ( I I I ) . The s i m i l a r i t i e s extend beyond t he general f o l d i n g of the back-bone, and many of the s i de chains are in the same p o s i t i o n s . The only c ons i de r a bl e d i f f e r e n c e s in s t r u c t u r e were found a t t he two ends of the B-chain, where t he r e s i d u e s in pig i n s u l i n are involved in hexamer for mat i on. Thei r s t r u c t u r a l a l t e r a t i o n in t he hag
f i s h i n s u l i n molecule i s t h e r e f o r e under s t andabl e. At the o t h e r end of t he B-chain r e s i due s B28-31 ™ hagf i sh ta k e s a d i f f e r e n t path in t he c r y s t a l s t r u c t u r e which probably r e f l e c t s the change in s t r u c t u r e in t h i s regi on.
In 2Zn pig i n s u l i n t he two molecules in t he dimer e x h i b i t d i f f e r e n c e s in conformation (38), and even more so in 4Zn i n s u l i n (44). The s t r u c t u r e of hagfi sh i n s u l i n i s c l o s e l y r e l a t e d t o pig i n s u l i n molecule 2 ( I I I ) . In f a c t , t he t e r t i a r y s t r u c t u r e of hagfi sh i n s u l i n was more s i m i l a r t o pig i n s u l i n molecule 2 than were pig i n s u l i n molecules 1 and 2. There a r e no publi shed data on hagf i sh i n s u l i n ' s behaviour in s o l u t i o n but the s t r i c t c ons er vat i on of the s t r u c t u r e of the dimer-forming r e s i due s in hagf i sh and pig 2Zn and 4Zn i n s u l i n s along with the obs er vat i on t h a t the d i m e n a t i on c ons t a nt of hagfi sh and pig i n s u l i n s are i n d i s t u i n g i s h a b l e wi t hi n one order of magnitude (B. Frank, unpublished), imply t h a t t h i s s t a b l e r egi on of the dimer i s p r e
served in s o l u t i o n , perhaps a l s o in the f r e e monomer (44).
Molecular e v o l ut i on of i n s u l i n
Since any b i o l o g i c a l funct i on i s a t l e a s t bi mol ecul ar i t follows t h a t i t s ev o l u t i o n i s a t l e a s t dual . I d e a l l y , t h e ev o l u t i o n of a hormone should i ncl ude a l s o t he e v o l ut i on of i t s e f f e c t o r ( s ) I n s u l i n r e c e p t o r s are consi dered t o have remained f u n c t i o n a l l y un a l t e r e d duri ng e v o l u t i o n , s i nce i n s u l i n s bind t o s pe c i e s d i f f e r e n t r e c e p t o r s in a way which r e f l e c t the o r i g i n of t he hormone r a t h e r than the r e c e p t o r ( 45) , with t he p o s s i b l e except i on of some i n s u l i n r e c e p t o r s from Hystricomorph r odent s (46). The cons er vat i on of t he behaviour of i n s u l i n r e c e p t o r s a ppl i e s t o i n s u l i n r e c e p t o r s from t he A t l a n t i c hagf i sh as well (47). Thus, hagf i s h e r y t h r o c y t e
i n s u l i n r e c e p t o r s show t i me - , t e mp e r a t u r e - , and pH dependence of i n s u l i n bindi ng t h a t ar e c h a r a c t e r i s t i c of ot he r i n s u l i n r e c e p t o r s . Moreover, t he h agf i s h e r y t h r o c y t e r e c e p t o r s (of unknown p hys i ol ogi cal s i g n i f i c a n c e ) have t he same a b s o l u t e a f f i n i t y and rank order of pr ef er ence f o r i n s u l i n s and i n s u l i n anal ogues, and the bindi ng a f f i n i t y of hagfi sh i n s u l i n was around 25%
(Fi g. 7, reproduced from ( 47) ) .
C h i c k e n In su lin P o r k In s u lin H a g f is h In s u lin
P o r k P r o in s u l in G u in e a P ig I n s u lin
O O P In s u lin
FIGURE 7 Binding of insulin and insulin a n a lo g s to hagfish erythrocytes. 12sl-pork insulin w a s incubated with hagfish erythrocytes in the a b s e n c e and p rese n c e of u nlabeled horm ones for 3 h at 15°C. In this experim ent, the maximum sp ecific binding (in the a b s e n c e of unlabeled hormone) w a s 2.6% of the total radioactivity. The amount of radioactivity specifically bound to the receptor in the p rese n c e of ea c h of the polypeptide horm ones w a s ex p re ssed a s a percent of maximum sp ecific binding. In Table 1, the results of this experim ent are com pared with five similar stu dies.
INSULIN CONCENTRATION (ng/ml)
Evolution, in t he Darwinian view, i s des cr i bed as p o s i t i v e nat ur al s e l e c t i o n . At t he mol ecul ar l e v e l , evo l u t i o n a r y change r e s u l t s from the spr eadi ng of accept ed f avour abl e mut ati ons in a populat ion (ctf. 48).
The f a c t t h a t , in a given famil y of p r o t e i n s t he number of s u b s t i t u t i o n s between two s pe c i e s i s a f unct i on of the known time of di ver gence, and i s independent of t he s peci es chosen suggest t h a t sequence changes are mostly n e u t r a l and f u n c t i o n a l l y i n s i g n i f i c a n t from t he s t a n d p o i n t of nat ur al s e l e c t i o n (49). This ne ut r a l mut ati on theor y impl i es t h a t molecules evolve i n d i v i d u a l l y a t a r a t e dependent on the f unc t i ona l co n s t r a i n t s of t he mol ecule, and once t he s t r u c t u r e and f unc t i on of a molecule are determined, e v ol ut i on a c t s mainly t o mai nt ai n them. Ohta (48) has suggested t h a t mol ecular e vol u
t i o n appears t o be a continuum where, a t one end, a r e l a t i v e l y l a r g e number of ne ut r a l mut ati ons occur wi t hout phenotypi c consequence. At t he o t h e r end, r e l a t i v e l y few mut ati ons a f f e c t i n g secondary and t e r t i a r y s t r u c t u r e s are found, on which Darwinian s e l e c t i v e pr e s s ur e s can a c t . The r a t e of i n s u l i n ev o l u t i o n is slow and uniform, except f o r t h a t of t he Hystricomorphs (38) and t h i s uniform r a t e of ev o l u t i o n has been taken as evidence in f avour of
ne u t r a l mut ati ons ( 40, 50) . The slow r a t e of i n s u l i n e v ol ut i on (about one t ent h of t h a t of t he C-pept ide) i n d i c a t e s t h a t the i n s u l i n molecule i s under heavy s t r u c t u r a l and f u n c t i o n a l c o n s t r a i n t s . On the ot he r hand Blundell and Wood have d es cr i bed i n s u l i n evol u t i o n as a Darwinian adapt i ve pr oces s , s uppor ti ng t h e i r ideas l a r g e l y on data from the Hystricomorph i n s u l i n s (10).
The s t r i c t p r e s e r v a t i o n of the t h r e e dimensional s t r u c t u r e of i n s u l i n in t he h a g f i s h , d e s p i t e changes in s t r u c t u r e and aggr egat ion (I and I I ) , favours t he idea t h a t e v o l ut i on has proceeded along a ne ut r a l p a t h, al though s u b t l e adapt i ve pr oces ses cannot be r ul e d out . In a d d i t i o n , the conser vat i on of r e c e p t o r bi ndi ng a f f i n i t y and b i o l o g i c a l a c t i v i t i e s (IV and VIII) s t r o n g ly s ugges t t h a t i n s u l i n and i t s pr e c ur s or s were s t r u c t u r a l l y and b i o l o g i c a l ly we l l - d e f i n e d a l r e a dy some 500 mi l l i o n year s ago. This al s o makes i t r eas onabl e t o assume t h a t the e n t i r e i n s u l i n pr oces si ng machinery ant edat ed t he appearance of t he v e r t e b r a t e s .
Degradation, r e c e p t o r bi ndi ng a f f i n i t y and potency of hagf i sh i n s u l i n in i s o l a t e d r a t f a t c e l l s
The degr adat i on of hagfi sh i n s u l i n , by a membrane a s s o c i a t e d f a t c e l l p r ot e a s e was s t udi e d in a c oncent r at ed f a t c e l l suspension (IV). The r e s u l t s i n d i c a t e t h a t both V a max and K of hagf i sh i n s u l i n degradat i on ar e about 10 m t imes lower than the corr espondi ng values f o r pig i n s u l i n . In d i l u t e f a t c e l l sus pensions , t he r e c e p t o r bindi ng a f f i n i t y of hagfi sh i n s u l i n i s 23%
of t h a t of pig i n s u l i n . The potency of hagfi sh i n s u l i n , with r e s p e c t to a c t i v a t i o n of 1 i p o ge ne s i s i s only about 5%. Hagfish i n s u l i n was t h e r e f o r e t he f i r s t p a r t i a l a n t a g o n i s t on the r e c e p t o r . On the whole c e l l i t e l i c i t s a f u l l response due t o the precence of spar e r e c e p t o r s (IV). The p a r t i a l antagonism demonstrates a d i s s o c i a t i o n between i n s u l i n bindi ng and e x p r e s s i on of b i o l o g i c a l a c t i v i t y , and a l s o t h a t the hagf i sh i n s u l i n - r e c e p t o r complex, a c t i v a t e s fewer glucose c a r r i e r s than t he pig i n s u l i n - r e c e p t o r complex.
However, Muggeo eX a t . could not reproduce t he observed dis cr epancy when t e s t i n g hagfi sh i n s u l i n (47). This prompted a r e - i n v e s t i g a t i o n using a
125 125
somewhat d i f f e r e n t approach (51). By using I - h a g f i s h and I - p i g i n s u l i n s i odi na t e d in p a r a l l e l , t h e i r binding in t r a c e equimolar concen
t r a t i o n (80 pM) to r a t f a t c e l l s was s t u d i e d . Of the added pig t r a c e r , 1,72%
was bound t o t he c e l l s and the corr esponding value f o r the hagf i sh i n s u l i n t r a c e r was 0.44%. The f r a c t i o n 0. 44/ 1. 72 (25.6%) is a d i r e c t measure of hag
f i s h i n s u l i n ' s r e l a t i v e r e c e p t o r a f f i n i t y . Despit e hagf i sh i n s u l i n ' s lower a f f i n i t y i t was e s t a b l i s h e d t h a t the d i s s o c i a t i o n r a t e co n s t a n t of hagfi sh i n s u l i n was about h a l f of t h a t of pig i n s u l i n ( Fi g. 8 reproduced from ( 5 1 ) ) . Hence, the a s s o c i a t i o n co n s t a n t can be c a l c u l a t e d t o be about 12% of t h a t
of pig i n s u l i n . I t was concluded t h a t Muggeo z t a t , (47) did not measure t he bi nding of hagf i sh i n s u l i n under s t eady s t a t e c o n d i t i o n s . Hagfish i n s u l i n ' s behavi our in s ever al t e s t systems i s shown in Table 1.
TABLE 1
Hagfish i n s u l i n : summary of b i o l o g i c a l behaviour r e l a t i v e to pig i n s u l i n
Rat f a t - c e l l s : References
Bi ol ogical a c t i v i t y 4-7% IV, 47
Receptor binding as:
I n h i b i t i o n of 125I - p i g i n s u l i n bi nding
125I - h a g f i s h i n s u l i n binding
Degradation as:
I n h i b i t i o n of 125I - pi g i n s u l i n degra d a t i o n by the membrane a s s o c i a t e d pr ot e a s e
Receptor mediated degr adat i on Rat 1i v e r cel Is :
Receptor bindi ng as:
I n h i b i t i o n of 125I - p i g i n s u l i n binding 3-7% 60, 61 Degradation as:
I n h i b i t i o n of 125I - p i g i n s u l i n degr a
dat i o n by the membrane a s s o c i a t e d 15nM(hagfish) 61
pr o t e a s e 120nM(pig)
Degradation v e l o c i t y 4% 60
Hagfish e r y t h r o c y t e s :
I n h i b i t i o n of 125I - p i g i n s u l i n bindi ng 25% 47 Human IM 9 lymphocytes:
I n h i b i t i o n of 125I - p i g i n s u l i n bindi ng 5-10% 47
125i - hagfis h i n s u l i n bindi ng 23% 51
A b i l i t y to induce negati ve c o - o p e r a t i v i t y 5% 39, 47
Other a nt a g o n i s t s have l a t e r been i d e n t i f i e d . Hence, porcupine i n s u l i n has been shown t o have a bindi ng a f f i n i t y and b i o l o g i c a l a c t i v i t y almost i d e n t i c a l t o hagf i sh i n s u l i n (52). S i mi l a r di s c r e p a n c i e s have been demon
s t r a t e d with a s y n t h e t i c A^-j - asparagi nami de-insul i n, which had 60% bindi ng a f f i n i t y and about 12% b i o l o g i c a l a c t i v i t y (53). Covalent l y l i nked i n s u l i n
- 12nM( hagfi s h ) IV 130nM(pig)
80-100% 51
23% IV
5-10% 47
25% 51
