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Isolation of a melanophore-stimulating peptide from pig pituitary gland
5()8
SHORT COMMUNICATIONS, PRt,;LIMIN.KR'r
NOTES
VI)L.
17 (~955)
Isolation of a melanophore-stimulating peptide from pig pituitary gland The melanoph()re e x p a n d i n g activity p r e s e n t in the p i t u i t a r y of vertebrates has received increasing interest durir, g recent years, which ill p a r t has been stimuhlted by a presumed relationship b e t w e e n this principle and the adrenocorticotrophic hormone. Investigations so far reported have been carried out with relatively i m p u r e preparations. Recently, however, LERNER AND I.EE 1 claim to have olltained an electrophoretically homogeneous substance t/ossessing a very high potency. By a milder procedure t h a n t h a t used by these authors, we have isolated a peptide which (lifters sutTstantially from theirs a and which has a lower molecular weight. Acetone-dried pig posterior lolTe 1)owder, kindly supplied by Dr. J. LENS, Organon, Holland, was purified according to the method of I~ANI)GREBE AND ~[[TCHEI.L 2. The initial material was of relatively low potency, containing a b o u t I. 5 " I n t e r n a t i o n a l " units/rag when tested in A'em)/)z~s laevis and the m e t h o d yiehled a p r e p a r a t i o n containing 3 o o 35 ° "].U."/mg. This preparation was frac1.40tionated by zone-electrophoresis in a vertical column using the a p p a r a t u s described by one of t ~. The t u b e (45 >: 3 cm) was packed with cellt:l~:~¢o p o w d e r ;~ccording to tile directions given l , y [PLODIN AND K U P K E 5. l'yridinium acetate solution was chosen as the conducting medium in order to easily recover the fractionated material in a salt-free condition. Very satisfactory frac tionation of 57 mg crude material was ohtained by using o.I molar pyridinium acetate (pH 4.8). A current of 3(7 mA wqs passed t h r o u g h tile colunnl for 60 hours with 175 V applied across tile electrodes. No loss of activity was obserxed but a considerable a m o u n t of inactive material left tile column in both directions. The active substance together with some inert material was removed from the column and collected in 4 lul porti(ms. The distribution of solid m a t t e r was estimated using the ninhydrin reaction and the portions A were pooled into larger fractions as indicated in Fig. I. In Fraction ll, 17 mg was recovered containing 80 % of tile total a m o u n t of activity =-=1 at/plied to tile c o l u n m ( a p t ) r o x i m a t e l y 90o l . ( I . / rag). This fraction was subjected to a new run under the same conditions as before except for Fig. ~. Distribution of n i n h y d r i n positive maionger d u r a t i o n (loo hours). A s h a r p peak con terial obtained in an electrophoretic r u n o n taining all tile activity lint only (75 *!4, of tile solid cohlnm as described in the text. Ordinate: OpInatter was obtained. The distribution of material tical density at 57(:7 nile. :\bscissa: Volume of within the peak was perfectly symmetrical acdisplaced solution from tile coluiun. The anodecording to ninhydrin, ultraviolet abs(~rtTtion (of direction corresponds to increasing displacement pyridin-free fractious) at 24 ° ]n/~ and 28o m H volumes. Tile arrow at A indicates the volume as well as amino acid distribution. This p r o d u c t of fluid between the filter plate at the b o t t o m contained 15no "l.U."/mg and is a b o u t lOOO and the starting positioi1 of the original zone. times as active as the original pig posterior lobe powder. By p r o p e r packing of the c o l u l n n , a s h a r p e r separation was achieved with this type of long duration zone-electrophoresis t h a n with electrophoresis on paper. 111 fact, substances which c a n n o t be distinguished by their electrophoretic b e h a v i o u r on paper can often be fractionated on satisfactorily prepared columns. The m o s t active p r e p a r a t i o n which a p p a r e n t l y is a peptide was s h o w n to be electrophoretically homogeneous at p H 3.5 (pyridinium formate o.~ molar with respect to formic acid) and at 7.0 (sodiun/ p h o s p h a t e u o.2). Due to the instability of the activity in alkaline solution, no run was performed at a higher p H t h a n 7.o. The isoelectric point of the peptide i n 0.05 molar p h o s p h a t e 6 solution w a s found to be 5.2 as determined by a procedure similar to t h a t by K U N K E L AND T I S E L I U S b u t using glucose and proline as reference substances to determine the electroosmotic flow. An a p p r o x i m a t e molecular weight of 3ooo was found by equilibriunl centrifugation, which also yielded additional evidence of the h o m o g e n e i t y of the preparation. A m o n g the p r o d u c t s of hydrolysis the following amino acids have been ideutified: aspartic and glutamic acids, serine, glycine, alanine, valine, proline, phenylalanine, tyrosine, histidine, lysine and arginine. Certain peptide linkages a p p e a r to be partieularly resistant tel acid hydrolysis. The peptide reacts with Ehrlich's reagent and ultra-
VOL. 1 7
(z(~55)
SHORT COMMUNICATIONS, PRELIMINARY NOTES
500
v i o l e t a b s o r p t i o n in a l k a l i n e s o l u t i o n r e v e a l s t h e p r e s e n c e of one mol e of b o t h t r y p t o p h a n e a nd t y r o s i n e . No s u l p h u r - c o n t a i n i n g a m i n o acid r e s i d u e s h a v e be e n found to be present. W h e n c o m p a r i n g t h e d a t a of LERNER AND LEE w i t h ours, t h e following s t r i k i n g differences are e n c o u n t e r e d : T h e isoelectric p o i n t of our p e p t i d e is a b o u t p H 5 as c o m p a r e d w i t h p H lO. 5 i i a n d is d e v o i d of ey stin e, leucine a n d t h r e o n i n e . [ t is difficult t o c o m p a r e t h e p o t e n c i e s of t h e t w o p r e p a r a t i o n s since t h e y were a s s a y e d differently. W e a s s a y e d our m a t e r i a l a c c o r d i n g t o t h e m e t h o d o f L A N D G R E B E AND \ ¥ A R I N G 8 t o a n a c c u r a c y
o f 10 0,/0 a g a i n s t a s t a n d a r d
material.
L E R N E R .AND LEE,
on t h e o t h e r hanlt, used a m u c h less a c c u r a t e m e t h o d of a s s a y b a s e d on t h e response of i s o l a t e d frog skin. M e t h o d s of this k i n d h a v e been a d e q u a t e l y c r i t i c i s e d elsewhereV, s. A p ossible e x p l a n a t i o n of t h e d i v e r g e n c e is, in our opinion, t h a t LERNER AND I~EE h a v e isolated a basic a g g r e g a t e of t h e a c t i v e p e p t i d e and i n e r t m a t e r i a l . \Ve h a v e o b s e r v e d a ba s i c c o m p o n e n t in tile m a t e r i a l e l u t e d from oxycellulose, t h e isoelectric p o i n t of w h i c h a p p e a r s to agree w i t h t h a t r e p o r t e d b y LERNER AND LEE. AS c o m p l e t e r e c o v e r y was found w h e n a s s a y i n g m a t e r i a l not i n c l u d i n g t h i s c o m p o n e n t its a c t i v i t y w a s n o t d e t e r m i n e d . A m o r e d e t a i l e d c o m m u n i c a t i o n c o n c e r n i n g t h e isola t i on a n d phys i c a l , c h e m i c a l a nd biological p r o p e r t i e s of t h e p e p t i d e will be p u b l i s h e d later.
Institute o/ Biochemistry, University o/ Uppsala, (Sweden)
J. PORATH P. R o o s F . W . LANDGREBE G . M . MITCHELL
Departme;zt o/ Pharmacology, Welsh National School o/ ~lledicine, Cardiff (Wales) J A. o F. 3 F. 4 j. 5 p. 6 H. 7 F. s H.
B. LERNER AND T. H. LEE, J. din. Chem. Soc., 77 (1955) lO66. \ ¥ . LANDGREBE AND G. M. MITCHELL, Quart. J. Exptl. Physiol., 39 (1954) I I . \V. I~ANDGREBE AND H. \VARING, Quart. J. ExptI. Physiol., 43 (1944) I. PORATH, dcta Chem. Scan&, 8 (I954) 1813. FLODIN AND D. \¥. KUPKE, to be p u b l i s h e d . ]KUNKEL AND A. TISELIUS, J. Gen. Physiol., 35 0 9 5 0 89. \V. LANDGI~EBE AND H. \ ¥ A R I N G , Quart..]. Exptl. Physiol., 31, ~941 31. \\;ARlYG AND F. W. LANDGREBE, Hormo~es, Vol. II. A c a d e m i c Press, N e w Y o r k (195o). R e c e i v e d M a y 241h, 1955
Influence de la ribonucl4ase sur la teneur en ad4nosinetriphosphate (ATP ) et la consommation d'oxyg~ne des amibes vivantes Un t r a v a i l rd cent de BRACHET2 a m o n t r 6 q u ' u n t r a i t e m e n t a p p r o p r i 6 5. la r i b o n u c l d a s e (RNase) p e r m e t de faire varier, p r e s q u e 5. v o l o n t 6 et d a n s le m 6 m e sens, la t e n e u r en acide r i b o n u c l d i q u e (ARN) et l ' i n c o r p o r a t i o n des acides a m i n d s d a n s les prot6ines, chez les a m i b e s v i v a n t e s (Amoeba proteus). No us a w m s cherch6 5. v o i r si la R N a s e modifie la t e n e u r en A TP e t la c o n s o m m a t i o n d ' o x y g6ne des a m i b e s ainsi traitdes, afin de pr6ciser si l ' e n z y n l e n ' a l t ~ r e p a s l ' a n a b o l i s m e p r o t 6 i q u e en i n h i b a n t la p r o d u c t i o n d'dnergie. L ' A T P a 616 dosd s u i v a n t la t e c h n i q u e de ~TREHLER 7, d a n s les c o n d i t i o n s ddc ri t e s r d c e n u n e n t p a r 1)RACHET a .
No us a v o n s fair a g i r la R N a s e cristallis6e (G.B.I.) p e n d a n t des t e m p s v a r i a b l e s , en vdrifiant c h a q u e lois l ' a c t i v i t 6 e n z y m a t i q u e des s o l u t i o n s p a r la m 6 t h o d e de KUNITZS; la c o n c e n t r a t i o n 6 t a i t de l ' o r d r e de o.i m g / m l . Les v a r i a t i o n s observ6es au cours de 8 s6ries d' e xp6ri e nc e s , p e n d a n t des t e m p s a l l a n t de 15 5. 18 h, ne s o n t p a s tr4,s i m p o r t a n t e s : la diffdrence e n t r e les m o y e n n e s e s t de l ' o r d r e de 25 o~) (T ab leau I). C e p e n d a n t , au b o u t d ' u n e d e m i - h e u r e 5. une heure, oi1 p e u t n o t e r une ldg6re a u g m e n t a t i o n de la t e n e u r en ATP, b i e n t 6 t s u i v i e d ' u n e c hut e . Si on les r e m e t d a n s leur milieu h a b i t u e l (liquide (te C h a l k l e y ) ou darts de I ' A R N (Schwarz 1. 4 m g / m l ) , les a m i b e s r e t r o u v e n t leur taux d'ATP normal. TABLEAU I T E N E U R MOYENNE EN A T P (/2g/Ioo amibes) DES AMIBES NORMALES ET TRAITEES X LA RNASE A mibes traitdes p a r l ' R N a s e A mibes normales
o.175
z5,
1/., h
~4 h
• h
z h ~,~
2 h
z8 h
+Chalkley
q ARN
o.186
o.196
o.220
0.205
o.172
o.17o
o.165
o.183
o.t77
* T o u t e s ces m e s u r e s o n t 6t6 f a i t e s a v e c le concours du Dr. M. ERRERA a u q u e l v o n t rues pl us vifs remerciements.
SHORT COMMUNICATIONS, PRt,;LIMIN.KR'r
NOTES
VI)L.
17 (~955)
Isolation of a melanophore-stimulating peptide from pig pituitary gland The melanoph()re e x p a n d i n g activity p r e s e n t in the p i t u i t a r y of vertebrates has received increasing interest durir, g recent years, which ill p a r t has been stimuhlted by a presumed relationship b e t w e e n this principle and the adrenocorticotrophic hormone. Investigations so far reported have been carried out with relatively i m p u r e preparations. Recently, however, LERNER AND I.EE 1 claim to have olltained an electrophoretically homogeneous substance t/ossessing a very high potency. By a milder procedure t h a n t h a t used by these authors, we have isolated a peptide which (lifters sutTstantially from theirs a and which has a lower molecular weight. Acetone-dried pig posterior lolTe 1)owder, kindly supplied by Dr. J. LENS, Organon, Holland, was purified according to the method of I~ANI)GREBE AND ~[[TCHEI.L 2. The initial material was of relatively low potency, containing a b o u t I. 5 " I n t e r n a t i o n a l " units/rag when tested in A'em)/)z~s laevis and the m e t h o d yiehled a p r e p a r a t i o n containing 3 o o 35 ° "].U."/mg. This preparation was frac1.40tionated by zone-electrophoresis in a vertical column using the a p p a r a t u s described by one of t ~. The t u b e (45 >: 3 cm) was packed with cellt:l~:~¢o p o w d e r ;~ccording to tile directions given l , y [PLODIN AND K U P K E 5. l'yridinium acetate solution was chosen as the conducting medium in order to easily recover the fractionated material in a salt-free condition. Very satisfactory frac tionation of 57 mg crude material was ohtained by using o.I molar pyridinium acetate (pH 4.8). A current of 3(7 mA wqs passed t h r o u g h tile colunnl for 60 hours with 175 V applied across tile electrodes. No loss of activity was obserxed but a considerable a m o u n t of inactive material left tile column in both directions. The active substance together with some inert material was removed from the column and collected in 4 lul porti(ms. The distribution of solid m a t t e r was estimated using the ninhydrin reaction and the portions A were pooled into larger fractions as indicated in Fig. I. In Fraction ll, 17 mg was recovered containing 80 % of tile total a m o u n t of activity =-=1 at/plied to tile c o l u n m ( a p t ) r o x i m a t e l y 90o l . ( I . / rag). This fraction was subjected to a new run under the same conditions as before except for Fig. ~. Distribution of n i n h y d r i n positive maionger d u r a t i o n (loo hours). A s h a r p peak con terial obtained in an electrophoretic r u n o n taining all tile activity lint only (75 *!4, of tile solid cohlnm as described in the text. Ordinate: OpInatter was obtained. The distribution of material tical density at 57(:7 nile. :\bscissa: Volume of within the peak was perfectly symmetrical acdisplaced solution from tile coluiun. The anodecording to ninhydrin, ultraviolet abs(~rtTtion (of direction corresponds to increasing displacement pyridin-free fractious) at 24 ° ]n/~ and 28o m H volumes. Tile arrow at A indicates the volume as well as amino acid distribution. This p r o d u c t of fluid between the filter plate at the b o t t o m contained 15no "l.U."/mg and is a b o u t lOOO and the starting positioi1 of the original zone. times as active as the original pig posterior lobe powder. By p r o p e r packing of the c o l u l n n , a s h a r p e r separation was achieved with this type of long duration zone-electrophoresis t h a n with electrophoresis on paper. 111 fact, substances which c a n n o t be distinguished by their electrophoretic b e h a v i o u r on paper can often be fractionated on satisfactorily prepared columns. The m o s t active p r e p a r a t i o n which a p p a r e n t l y is a peptide was s h o w n to be electrophoretically homogeneous at p H 3.5 (pyridinium formate o.~ molar with respect to formic acid) and at 7.0 (sodiun/ p h o s p h a t e u o.2). Due to the instability of the activity in alkaline solution, no run was performed at a higher p H t h a n 7.o. The isoelectric point of the peptide i n 0.05 molar p h o s p h a t e 6 solution w a s found to be 5.2 as determined by a procedure similar to t h a t by K U N K E L AND T I S E L I U S b u t using glucose and proline as reference substances to determine the electroosmotic flow. An a p p r o x i m a t e molecular weight of 3ooo was found by equilibriunl centrifugation, which also yielded additional evidence of the h o m o g e n e i t y of the preparation. A m o n g the p r o d u c t s of hydrolysis the following amino acids have been ideutified: aspartic and glutamic acids, serine, glycine, alanine, valine, proline, phenylalanine, tyrosine, histidine, lysine and arginine. Certain peptide linkages a p p e a r to be partieularly resistant tel acid hydrolysis. The peptide reacts with Ehrlich's reagent and ultra-
VOL. 1 7
(z(~55)
SHORT COMMUNICATIONS, PRELIMINARY NOTES
500
v i o l e t a b s o r p t i o n in a l k a l i n e s o l u t i o n r e v e a l s t h e p r e s e n c e of one mol e of b o t h t r y p t o p h a n e a nd t y r o s i n e . No s u l p h u r - c o n t a i n i n g a m i n o acid r e s i d u e s h a v e be e n found to be present. W h e n c o m p a r i n g t h e d a t a of LERNER AND LEE w i t h ours, t h e following s t r i k i n g differences are e n c o u n t e r e d : T h e isoelectric p o i n t of our p e p t i d e is a b o u t p H 5 as c o m p a r e d w i t h p H lO. 5 i i a n d is d e v o i d of ey stin e, leucine a n d t h r e o n i n e . [ t is difficult t o c o m p a r e t h e p o t e n c i e s of t h e t w o p r e p a r a t i o n s since t h e y were a s s a y e d differently. W e a s s a y e d our m a t e r i a l a c c o r d i n g t o t h e m e t h o d o f L A N D G R E B E AND \ ¥ A R I N G 8 t o a n a c c u r a c y
o f 10 0,/0 a g a i n s t a s t a n d a r d
material.
L E R N E R .AND LEE,
on t h e o t h e r hanlt, used a m u c h less a c c u r a t e m e t h o d of a s s a y b a s e d on t h e response of i s o l a t e d frog skin. M e t h o d s of this k i n d h a v e been a d e q u a t e l y c r i t i c i s e d elsewhereV, s. A p ossible e x p l a n a t i o n of t h e d i v e r g e n c e is, in our opinion, t h a t LERNER AND I~EE h a v e isolated a basic a g g r e g a t e of t h e a c t i v e p e p t i d e and i n e r t m a t e r i a l . \Ve h a v e o b s e r v e d a ba s i c c o m p o n e n t in tile m a t e r i a l e l u t e d from oxycellulose, t h e isoelectric p o i n t of w h i c h a p p e a r s to agree w i t h t h a t r e p o r t e d b y LERNER AND LEE. AS c o m p l e t e r e c o v e r y was found w h e n a s s a y i n g m a t e r i a l not i n c l u d i n g t h i s c o m p o n e n t its a c t i v i t y w a s n o t d e t e r m i n e d . A m o r e d e t a i l e d c o m m u n i c a t i o n c o n c e r n i n g t h e isola t i on a n d phys i c a l , c h e m i c a l a nd biological p r o p e r t i e s of t h e p e p t i d e will be p u b l i s h e d later.
Institute o/ Biochemistry, University o/ Uppsala, (Sweden)
J. PORATH P. R o o s F . W . LANDGREBE G . M . MITCHELL
Departme;zt o/ Pharmacology, Welsh National School o/ ~lledicine, Cardiff (Wales) J A. o F. 3 F. 4 j. 5 p. 6 H. 7 F. s H.
B. LERNER AND T. H. LEE, J. din. Chem. Soc., 77 (1955) lO66. \ ¥ . LANDGREBE AND G. M. MITCHELL, Quart. J. Exptl. Physiol., 39 (1954) I I . \V. I~ANDGREBE AND H. \VARING, Quart. J. ExptI. Physiol., 43 (1944) I. PORATH, dcta Chem. Scan&, 8 (I954) 1813. FLODIN AND D. \¥. KUPKE, to be p u b l i s h e d . ]KUNKEL AND A. TISELIUS, J. Gen. Physiol., 35 0 9 5 0 89. \V. LANDGI~EBE AND H. \ ¥ A R I N G , Quart..]. Exptl. Physiol., 31, ~941 31. \\;ARlYG AND F. W. LANDGREBE, Hormo~es, Vol. II. A c a d e m i c Press, N e w Y o r k (195o). R e c e i v e d M a y 241h, 1955
Influence de la ribonucl4ase sur la teneur en ad4nosinetriphosphate (ATP ) et la consommation d'oxyg~ne des amibes vivantes Un t r a v a i l rd cent de BRACHET2 a m o n t r 6 q u ' u n t r a i t e m e n t a p p r o p r i 6 5. la r i b o n u c l d a s e (RNase) p e r m e t de faire varier, p r e s q u e 5. v o l o n t 6 et d a n s le m 6 m e sens, la t e n e u r en acide r i b o n u c l d i q u e (ARN) et l ' i n c o r p o r a t i o n des acides a m i n d s d a n s les prot6ines, chez les a m i b e s v i v a n t e s (Amoeba proteus). No us a w m s cherch6 5. v o i r si la R N a s e modifie la t e n e u r en A TP e t la c o n s o m m a t i o n d ' o x y g6ne des a m i b e s ainsi traitdes, afin de pr6ciser si l ' e n z y n l e n ' a l t ~ r e p a s l ' a n a b o l i s m e p r o t 6 i q u e en i n h i b a n t la p r o d u c t i o n d'dnergie. L ' A T P a 616 dosd s u i v a n t la t e c h n i q u e de ~TREHLER 7, d a n s les c o n d i t i o n s ddc ri t e s r d c e n u n e n t p a r 1)RACHET a .
No us a v o n s fair a g i r la R N a s e cristallis6e (G.B.I.) p e n d a n t des t e m p s v a r i a b l e s , en vdrifiant c h a q u e lois l ' a c t i v i t 6 e n z y m a t i q u e des s o l u t i o n s p a r la m 6 t h o d e de KUNITZS; la c o n c e n t r a t i o n 6 t a i t de l ' o r d r e de o.i m g / m l . Les v a r i a t i o n s observ6es au cours de 8 s6ries d' e xp6ri e nc e s , p e n d a n t des t e m p s a l l a n t de 15 5. 18 h, ne s o n t p a s tr4,s i m p o r t a n t e s : la diffdrence e n t r e les m o y e n n e s e s t de l ' o r d r e de 25 o~) (T ab leau I). C e p e n d a n t , au b o u t d ' u n e d e m i - h e u r e 5. une heure, oi1 p e u t n o t e r une ldg6re a u g m e n t a t i o n de la t e n e u r en ATP, b i e n t 6 t s u i v i e d ' u n e c hut e . Si on les r e m e t d a n s leur milieu h a b i t u e l (liquide (te C h a l k l e y ) ou darts de I ' A R N (Schwarz 1. 4 m g / m l ) , les a m i b e s r e t r o u v e n t leur taux d'ATP normal. TABLEAU I T E N E U R MOYENNE EN A T P (/2g/Ioo amibes) DES AMIBES NORMALES ET TRAITEES X LA RNASE A mibes traitdes p a r l ' R N a s e A mibes normales
o.175
z5,
1/., h
~4 h
• h
z h ~,~
2 h
z8 h
+Chalkley
q ARN
o.186
o.196
o.220
0.205
o.172
o.17o
o.165
o.183
o.t77
* T o u t e s ces m e s u r e s o n t 6t6 f a i t e s a v e c le concours du Dr. M. ERRERA a u q u e l v o n t rues pl us vifs remerciements.