In vitro lipolysis of proopiocortin peptides

Life Sciences, Vol. 33, Sup. I, 1983, pp. 747-750 Printed in the U.S.A.

IN V I T R O

LIPOLYSIS

OF P R O O P I O C O R T I N

W.O.Richter

Pergamon Press

PEPTIDES x

and P . S c h w a n d t

D e p a r t m e n t of Internal M e d i c i n e sity of Munich, M a r c h i o n i n i s t r .

II, K l i n i k u m Grosshadern, Univer15, D-8ooo M u n i c h 7o, W - G e r m a n y

(Received in final form June 26, 1983) SUMMARY Since there is a c c u m u l a t i n g evidence for m e t a b o l i c functions of p r o o p i o c o r t i n peptides, we i n v e s t i g a t e d 42 p a r t i a l sequences for their lipolytic a c t i v i t y in i s o l a t e d rabbit fat cells. 4 lipolytic active centers can be suggested: in the N - t e r m i n a l part of p r o o p i o c o r t i n (not gamma- and delta-MSH), in the N - t e r m i n a l region of A C T H (ACTH 4-1o), in the B-MSH sequence of ~ - l i p o t r o p i n and in the C - t e r m i n a l region of B-endorphin.

The lipolytic a c t i v i t y of p r o o p i o c o r t i n p e p t i d e s resides partly in the h e p t a p e p t i d e sequence, M e t - G l u - H i s - P h e - A r g - T r p - G l y (I), common to A C T H (positions 4-1o) and ~ - l i p o t r o p i n (B-LPH h 45-51, ~D-LPH 47-53). Moreover, lipolytic a c t i v i t y was o b s e r v e ~ for humah and porcine B - e n d o r p h i n (2,3,4). In the N - t e r m i n a l part of p r o o p i o c o r t i n - t r p - r i c h peptide-(5) the two additional amino acid sequences gamma- and d e l t a - M S H were identified (6). Their amino acid sequences are similar to the common h e p t a p e p t i d e m e n t i o n e d above. To e v a l u a t e the lipolytic active centers of p r o o p i o c o r t i n we investigated the effects of 42 fragments of this p r e c u r s o r p r o t e i n from d i f f e r e n t species on lipid m o b i l i z a t i o n in i s o l a t e d rabbit adipocytes. MATERIAL

AND M E T H O D S

The p e p t i d e s and peptide sequences tested for lipolytic a c t i v i t y are shown with their origin in table I. Bovine a l b u m i n fraction V was p u r c h a s e d from Roth, Karlsruhe, WGermany, c o l l a g e n a s e CLS II from W o r t h i n g t o n (Seramed, Munich, WGermany). Bovine serum a l b u m i n fraction V was d e f a t t e d by the m e t h o d of Chen (7). P e r i r e n a l adipose tissue from female rabbits (white N e u s e e l a n d e r , 4-5 kg body weight) was o b t a i n e d after 12 hours of starvation. Isolated fat cells were p r e p a r e d by the m e t h o d of Rodbell (8).Inc u b a t i o n s were p e r f o r m e d in triplicate, using 7o oo0-9o ooo cells per tube, for 1 hour in K R B - b u f f e r pH 7.4 w i t h 5 % BSA V under continuous shaking. A f t e r removal of the fat cell layer the s u b n a t a n t was deep frozen at -35°C until glycerol analysis. G l y c e r o l was det e r m i n e d e n z y m a t i c a l l y by an a u t o m a t i c p r o c e d u r e as d e s c r i b e d prev i o u s l y (9). M i n i m a l e f f e c t i v e c o n c e n t r a t i o n s were c a l c u l a t e d from the log dose response curves. XSupported

by Deutsche

Forschungsgemeinschaft, SFB o2o7-LP 0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.

3o

748

Lipolysis by Proopiocortin Peptides

TABLE Lipolytic

Activity

Vol. 33, Sup. I, 1983

I

of P r o o p i o c o r t i n

Fragments

in R a b b i t

Adipocytes

minimal effective concentration a)

N - t e r m i n a l p a r t of p r o o p i o c o r t i n Trp-rich peptide ¥-MSH -MSH ACTH ACTH 1-39 ACTH p 1-24 ACTH~ 1-24 ACTH n 1-13 = ~ - M S H ACTH p I-Io ACTH~ I-4 ACTH~ 4-11

b)

ACTH~ 4-1o

ACTH h 18-39 B-lipotropin ~-LPH 1-89 ~ - L P H _ 1-91

c)

~-LPH~ 1-79

~-LPH I-Io ~-LPH 1-38 ~-LPH 37-58 = ~ - M S H ~-LPH 39-45 ~-LPH 41-58 ~-LPH 47-53 B - L P H p 61-65 B-LPH 61-76 = ~ - e n d o r p h i n B-LPH 61-77 = ¥ - e n d o r p h i n ~ - L P H p 61-91 = B D e n d o r p h i n ~ - L P H h 59-89 ~endorphin ~-LPH 59-87 B - L P H h 59-63 - 74-89 B-LPHh[D-ala6o,met63174-89 ~ - L P H h 64-89 B - L P H h 64-87 ~ - L P H h 85-89 ~ - L P H p 88-91 ~ - L P H ~ 89-91

B-LPH~ 86-89 ~-LPH o B-LPH B-LPH~ ~-LPH~ B-LPH o B-LPH o ~-LPH o B-LPH o

A B C D E F G H

= = = = = = = =

1-34 49-57 58-59 60-67 68-72 73-76 50-52 53-62

We t h a n k Purchased Purchased Purchased Prepared We t h a n k Purchased We t h a n k

A S

Io-6M

B

C B D B D m D B D

Io-9M 1o-WM Io-9M Io-9M Io-6M

E E F S E D G

Io-9M 5 x Io-I°M

B

1o

B

Io-6M

Io-6M Io-6M

~-9 Io M ~-9 M

c c

B E B

Io-9M Io-8M

H

H D H

H H F n F F F F F F F F

Io - 7 M

1¢o-8M ¢ ¢ ¢ ¢ ¢ ¢ ¢ ¢ ~ ¢ ¢ ¢

Dr. H a k a n s o n , L u n d , S w e d e n for the g e n e r o u s g i f t from Bachem,Burgdorf,Switzerland from Serva,Heidelberg,W-Germany from Peninsula,San Francisco,USA as d e s c r i b e d p r e v i o u s l y (2,4,13) Dr. N a u d ~ , P o r t E l i z a b e t h , S - A f r i c a , f o r the g e n e r o u s from Paesel,Frankfurt,W-Germany Dr.Morley,ICI,Cheshire,U.K.,for the g e n e r o u s g i f t

gif~

FIGURE

I

of Tested Peptides Containinq

2

O-B-LPH 35-46

•Ala-Pro-Leu-Lys-Asp

residues:

human B-lipotropin 89 (Io) porcine ~-lipotropin 91 (11) ostrich B-lipotropin 79 (12)

= ~-LPH h = B-LPHB-LPH~o

ostrich

Ser-G•n-Thr-Pr•-Leu-va•-Thr-Leu-Phe-Lys-Asn-A•a-I•e-va•-Lys-Asn-A•a-His-Lys-Lys-G•y-G•n-•H

G•y-Arg-A•a-Pr•-Leu-Va•-Thr-Leu-Phe-Lys-Asn-A•a-I•e-Va•-Lys-Ser-A•a-Tyr-Lys-Lys-G•y-G•n-•H

Total

porcine

human

¥-MSH

delta-MSH

ACTH 4-11

•Arg-Phe-Gly

.Lys

~-MSH ACTH I-Io

.Lys-Pro-Val

ACTH 4-Io

p-B-MSH

Ser-Pro-Pro-Lys-Asp

C-terminal Amino Acid Sequences of PorcinerHuman and Ostrich ~-lipotropin 7o 75 80 85 89 Ser-G•n-Thr-Pr•-Leu-Va•-Thr-Leu-Phe-Lys-Asn-A•a-I•e-I•e-Lys-Asn-A•a-Tyr-Lys-Lys-G•y-G•u-•H

FIGURE

Met-Arq-His-Phe-Arg-Trp-Gln

Ser. -Met-Glu-Val-Arq-Gly-Trp Tyr-Val. - M e t - ~ - H i s - P h e - A r g - T r p - A s p

Met-Glu-His-Phe-Arg-Trp-Gly

Met-Glu-His-Phe-Arg-Trp-Gly

Ser-Tyr-Ser •.Met-Glu-His-Phe-Arg-Trp-Gly Ser-Tyr-Ser. -Met-Glu-His-Phe-Arg-Trp-Gly

h-B-MSH

or Related Sequences

,Ser-Pro-Pro-Lys-Asp

the Common Heptapeptide

Ala-Glu-Lys-Lys-Asp-Glu-Gly-Pro-Tyr-Arg i.Met-Glu-His-Phe-Arg-Trp-Gly Asp-Glu-Gly-Pro-Tyr-Lys" -Met-Glu-His-Phe-Arg-Trp-Gly

Amino Acid Sequences

%o

D G~

I=l

o o o

o o m:l

ml

~.

O

OO

H

0

750

Lipolysis by Proopiocortin Peptides

RESULTS

Vol. 33, Sup. I, 1983

AND D I S C U S S I O N

The m i n i m a l e f f e c t i v e c o n c e n t r a t i o n s of the tested 42 partial sequences of p r o o p i o c o r t i n from d i f f e r e n t species are s u m m a r i z e d in table I. The h i g h e s t tested c o n c e n t r a t i o n was Io-6M for all fragments. T r p - r i c h peptide, the N - t e r m i n a l part of p r o o p i o c o r t i n , had lipolytical a c t i v i t y in a m i n i m a l e f f e c t i v e c o n c e n t r a t i o n of Io-6M, while its partial sequences gamma- and d e l t a - M S H were not active, suggesting that the t r p - r i c h p e p t i d e ' s lipolytic a c t i v i t y is not mediated by these two sequences to the common h e p t a p e p t i d e (figure I). A second lipolytic active core is located in the N - t e r m i n a l part of ACTH. L e n g t h e n i n g of A C T H 4-Io by an a d d i t i o n a l lysin residue to A C T H 4-11 and e l o n g a t i o n to A C T H I-Io did not increase the minimal e f f e c t i v e c o n c e n t r a t i o n of Io-6M. However, the l e n g t h e n i n g to A C T H 1-13 (alpha-MSH) increases the lipolytie a c t i v i t y (Io-9M). F u r t h e r e l o n g a t i o n (ACTH 1-24, ACTH 1-39) did not result in an increase of the lipolytic activity. In human and porcine ~ - l i p o t r o p i n two lipolytic centers were described previously: in the ~-MSH sequence r e s p e c t i v e l y in the Cterminal p o r t i o n of B - e n d o r p h i n (4). The two d i f f e r e n t residues in the common h e p t a p e p t i d e sequence of ostrich ~-LPH (figure I) might be one reason for the m i s s i n g lipolytic activity. A further explanation m i g h t come from the serine residue at p o s i t i o n 73 (figure 2) The d i f f e r e n c e s in the C - t e r m i n a l part might be r e s p o n s i b l e for the d i f f e r e n c e s in the lipolytic potency of p o r c i n e and human B-endorphin. The importance of the C - t e r m i n a l amino acid residues is s u p p o r t e d by the finding that [D-ala6o , met63 , 59-63]-74-87 is ineffective, while [59-63]-74-89 shows lipolytic activity. In conclusion, 4 lipolytic active centers can be found in the proo p i o c o r t i n - one in the N - t e r m i n a l part (but not gamma- or deltaMSH), the second in A C T H 4-1o, the third in the B-MSH region of ~lipotropin and the forth in the C - t e r m i n a l part of B-endorphin. The findings w i t h endorphins and ~ - l i p o t r o p i n s from d i f f e r e n t species suggest a role of p r o t e a s e s on their lipolytic function. REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. Io. 11. 12. 13.

M.W.DRAPER, R . B . M E R R I F I E L D and M.A.RIZACK, J.Med. Chem. 1 6 , 1 3 2 6 133o (1973) P.SCHWANDT, W . R I C H T E R and J.WILKENING, FEBS Lett.loo, 360-362 (1979) E . J E A N - B A P T I S T E and M.A.RIZACK, Life Sci.27, 135-141 (198o) P.SCHWANDT, W . R I C H T E R and J.S.MORLEY, Neur-~peptides i, 211216 (1981) R.HAKANSON, R.EKMAN, F . S U N D L E R and R.NILSSON, Nature 283, 78979o (198o) S.NAKANISHI, A.INOUE, T.KITA, M.NAKAMURA, A.C.Y.CHANG, S.N. C H O H E N and S.NUMA, Nature 278, 423 (1979) R.F.CHEN, J.Biol. Chem. 242, 173-181 (1967) M.RODBELL, J.Biol.Chem. 239, 375-380 (1964) P.SCHWANDT, W . O . R I C H T E R and N.KRIEGISCH, Clin. Chem. 27, 512513 (1981) C.H.LI and D.CHUNG, Int. J . P e p t i d e Prot. Res. 17, 131-142 (1981) L.GRAF, E.BARAT, G.CSEH and M.SAJGO, Biochim.--Biophys.Acta 229, 276-278 (1971) R.J.NAUDE, D.CHUNG, C.H.LI, W.OELOFSEN, Int. J.Pept. Prot. Res. I_88, 138-147 (1981) P . S C H W A N D T and W.RICHTER, J.Clin. Chem.Clin. Biochem. 18,736(1980)