Sodium hydroxide treatment of l -histidyl-l -phenylalanyl-l -arginyl-l -tryptophylglycine and its potentiated melanocyte-stimulating activity in vitro

337

BIOCHIMICA ET BIOPHYSICA ACTA

BBA

25464

SODIUM H Y D R O X I D E T R E A T M E N T OF L - H I S T I D Y L - L - P H E N Y L A L A N Y L L - A R G I N Y L - L - T R Y P T O P H Y L G L Y C I N E AND ITS P O T E N T I A T E D MELANOCYTE-STIMULATING ACTIVITY I N V I T R O K. HANO, M. KOIDA, H. YAJIMA, K. K U B O AND T. OSHIMA

Faculty of Pharmaceutical Sciences, Osaka University, Osaka (Japan) and Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto (Japan) (Received J u l y i3th, 1965)

SUMMARY

Treatment of L-histidyl-L-phenylalanyl-L-arginyl-L-tryptophylglycine, a common fragment of ~- and fl-MSH and ACTH, with o.I N sodium hydroxide at IOO° for IO min brought about a 4o-fold increase in the MSH activity on frog-skin melanocytes in vitro. Chemical and enzymatic examinations of this treated peptide showed that one quarter of the arginine was converted to ornithine and four amino acid residues: histidine, phenylalanine, arginine and tryptophan, were racemized. As far as the MSH activity in vitro is concerned, potentiation is the only phenomenon which was observed. Neither retarded nor prolonged effects of its action could be detected in this product. When the relative potencies of the synthetic stereoisomers of the pentapeptide were measured against the darkening value produced b y a constant amount of the all-L pentapeptide, L-histidyl-D-phenyalanyl-L-arginyl-L-tryptophylglycine and L-histidyl-L-phenylalanyl-L-arginyl-D-tryptophylglycine exhibited some 16o and 20 times higher activity than that of the all-L pentapeptide respectively. Based on these observations, the relationship between structure and the potentiated activity of the alkali-treated all-L pentapeptide is discussed. A possible explanation for the phenomenon of appearance or disappearance of the potentiated activity in alkali-treated ACTH and MSH's is proposed.

INTRODUCTION

Alkali-induced racemization has recently been examined in ~-MSH1, 2, fl-MSH 3 and ACTH 4. Two groups of investigators, LEE et al.1, 2 and LI et al.3, 4 arrived at the same conclusion that alkali treatment of these peptides brought about the modification in their melanocyte-stimulating properties. Two types of modified MSH activity towards frog melanocytes in vivo have been mentioned by other authorsS, 6 for the alkali-treated pituitary extract. But only one, namely the prolongation effect, increased time of darkening of frog-skin, was produced b y the alkali-treated peptide hormones, ~-MSH1, 2, fl-MSH 3 and ACTH 4. The other property, the so-called potentiation, an increased activity, was observed only in alkali-treated ACTH1,4,7, s. A third effect, namely the retardation effect, the delayed onset of the darkening action, Biochim. Biophys. Acta, 115 (1966) 337-344

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K. HANO, M. KOIDA, H. YAJIMA, K. KUBO, T. OSHIMA

was reported by LEE AXD B.-JANuSCR 2 in the alkali-treated a-MSH system in vitro. In 1959, LI 9 reported briefly that alkali treatment of L-histidyl-L-phenylalanylL-arginyl-L-tryptophylglycine, which represents the common sequence of these hormones, resulted in prolongation of its MSH activity. It was speculated that either the conversion of arginine to ornithine, or the racemization of the phenylalanine residue, or both, might have taken place during the alkali treatment of this fragment. Subsequently, three model pentapeptides, L-histidyl-L-phenylalanyl-L-ornithyl-Ltryptophylglycine ~°, L-histidyl-D-phenylalanyl-L-ornithyl-L-tryptophylglycine~° and L-histidyl-D-phenylalanyl-L-arginyl-L-tryptophylglycine ~1, were synthesized, and it was reported that the last two peptides containing a D-phenylalanyl residue showed the characteristic prolongation effect. Recently PICKERIX6 AND L112 revised their view by reporting that the alkali-treated pentapeptide exhibited no prolongation phenomenon. We have treated our synthetic all-L pentapeptide with sodium hydroxide under the identical conditions described by LEE et al."- and observed, after careful comparison of the MSH activities of the original and the treated sample, that the alkali-treated sample exhibited considerable potentiation of its activity in experiments in vitro. However, we observed no prolongation or retardation phenomenon in this treated fragment. Since our observation on this alkali-treated pentapeptide is considerably different from that reported by PICKERING aND L112, and no evidence for the chemical changes which could have taken place after the alkali treatment of this fragment has been reported by these authors, we have made a detailed examination of the alkalitreated all-L pentapeptide by using the enzymatic technique. The results are reported here, and a possible relationship between structure and potentiated activity of the alkali-treated pentapeptide is discussed. MATERIAL AND METHODS

Peptide All-c pentapeptide was synthesized according to the method described by HOFMANN ,aND LANDEla,14. The final product was purified on a CM-cellulose (BIO.RAD, Cellex-CM) 15 column using o.I M pyridine acetate buffer (pH 5.o) as an eluent. After lyophilization, a white fluffy powder was obtained, [e!D7 --11.6 ° (c O.5 in I N hydrochloric acid) (Ref. 13, [~]~' -11.8 ° in I N hydrochloric acid), single ninhydrin, Pauly, Sakaguchi and Ehrlich positive spot, RF o.51 (in the system of PARTRIDGE1G),Rphe 1.2 (in the system of 2-butanol-ammonia17). Amino acid ratios in an acid hydrolysate were histidine/phenylalanine/arginine/glycine (1.oo:o.99: I.OO: I.Oi). The result of leucine aminopeptidase (EC 3.4.1.1) digestion was histidine/phenylalanine/arginine/ tryptophan/glycine (i.oi : I.OI : 0.98 : 0.95 : I.OO).

Bioassay The method employed was essentially the same as that described by LERNER AXD WRmHT 18. In each experiment, four male frogs (Rana nigromaculata H.) were used.

Procedure .for preliminary sodium hydroxide treatment in a

The all-L pentapeptide (60 mg) in o.I N sodium hydroxide (30 ml) was heated boiling-water bath. Periodically, 5.0 ml were removed from the solution. Each

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ALKALI TREATMENT OF AN ACTIVE FRAGMENT OF a-MSH

339

fraction was cooled to room temperature, and the solution was neutralized to pH 7.37.4 by the addition of o.I N hydrochloric acid. Prior to heating, an aliquot (5 ml) of the original solution was neutralized and set aside as control. This control sample was found to have the same activity as that of the starting material which was not exposed to alkali.

Purification procedure of alkali-treated all-L pentapeptide The above preliminary study showed that heating for IO rain increased the activity 4o-fold, and further treatment for I h did not change this highly enhanced activity. The all-L pentapeptide (200 rag) in o.I N sodium hydroxide (20 ml) was heated in a boiling-water bath for 30 rain. The solution was neutralized with I N hydrochloric acid (2 ml). When an aliquot of this neutral solution was examined b y paper chromatography, no extra spot was detected although a slight tailing of the spot corresponding to the starting material was observed. This solution, after dilution with water (150 ml), was applied to a CM-cellulose 15 column (1. 5 cm × 20 cm) which was eluted with the following pH 5.0 pyridine acetate buffers: 0.05 M (250 ml) and o.I M (75 ° ml). Individual fractions of 15 ml each were collected with an automatic fraction collector at a flow rate of 5-6 ml/min. Measurement of absorbance at 28o m ~ served to locate the peptide. A single peak having a little shoulder was present in o.I M buffer (Tubes 35-65). This fraction was pooled, and the solvent was removed b y evaporation. The residue was lyophilized; yield 177 nag ~c~]~7 --4.4 ° (c 0.5 in i N hydrochloric acid), RF 0.52 in the system of PARTRIDGETM, Rphe I.O in the system of 2 - b u t a n o l - a m m o n i a 17 with slight tailing in each system. The MSH activity of this product was found to be forty times that of the starting material.

Acid hydrolysis Acid hydrolysis was performed in a sealed Pyrex tube with 5.7 N hydrochloric acid (b.p. IIO°), and amino acid ratios in the hydrolysate were determined with a Hitachi Amino Acid Analyzer KLA-2 according to the method of MOORE et alY"~.

Enzymatic procedure Amino acid ratios in enzymatic hydrolysates were also determined with an automatic amino acid analyzer, Hitachi KLA-2. Leucine aminopeptidase was prepared according to the method described b y SPACKMAN et al. ~°. Leucine aminopeptidase digestion was performed as described by HOFMANN AND YAJIMA21. Trypsin (EC 3.4.4.4) and ~-chymotrypsin (EC 3.4.4-5) digestions were performed according to the procedure described by LEE AND B.-JANuSCa 2. L-Amino acid oxidase (EC 1.4.3.2 ) and D-amino acid oxidase (EC 1.4.3.3) digestions were conducted according to the procedure described by LEE AND B.-JANUSCH~.

Microbiological assay of L-histidiue L-Histidine was determined quantitatively with Leuconostoc mesenteroides P-60. The method employed was essentially the same as that described b y BLOCK22.

Paper chromatography Toyo filter paper No, 51 was used. Descending chromatography was employed with the system of PARTRIDGETM, and with 2-butanol-3 % ammonia 17.

Biochim. Biophys. Acta, 115 (1966) 337-344

34 °

K. HANO, M. KOIDA, H. YAJIMA, K. KUBO, T. OSHIMA

RESULTS

Changes in melanocyte-stimulating activity Fig. I indicates the effect of heating, for IO, 2o, 3o and 6o min, on the melanocyte-stimulating activity of the all-L pentapeptide. Heating for io rain enhanced its activity 4o-fold, and this level of activity was maintained for a period of 60 rain. The pattern of darkening, caused by the purified sample of the alkali-treated all-L pentapeptide, and its behaviour towards repeated washing with Ringer solution, are illustrated in Fig. 2. When equipotent doses of the alkali-treated all-L pentapeptide (1. 5/~g/ml) and the untreated all-L pentapeptide (60/zg/ml) were used, no significant difference was observed between the time response curves produced by both peptides. The above observations demonstrate that the action of the treated all-L pentapeptide neither retards nor prolongs the activity in vitro. As shown in Fig. 3, when I)-histidyl-D-phenylalanyl-D-arginyl-D-tryptophylglycine (all-D) which acts as a specific inhibitor against the all-L pentapeptide was used, a similar type of lightening was observed on the skin predarkened by the alkali-treated all-L pentapeptide. Changes in chemical structure Examination of the reaction mixture of the alkali-treated pentapeptide by paper chromatography showed that no cleavage of its peptide bonds occurred during this treatment. The rotation value of the alkali-treated sample was considerably lower than that of the starting material. The results of chemical, enzymatic and microbiological analysis are summarized in Table I. Acid hydrolysis of the treated all-L pentapeptide gave satisfactory recovery of the constituent amino acids in the ratios predicted by theory, except arginine. Ornithine, which was derived from arginine, was present in the hydrolysate. Although the untreated all-L pentapeptide was cornA ~

oC

e

1oo c

/

4o 2, 3o o

~20 o

o

0)

.c_ 50 L.

!

lO

2;

3'0 ('(

6'0

o

3'0

9'0

Time (rain) Time (rain) Fig. I. The effect on the melanocyte-stimulating activity of the all-L p e n t a p e p t i d e of the alkaliheat t r e a t m e n t for several time intervals. On the ordinate the activity of the u n t r e a t e d all-L pentapeptide was taken as I.O. Fig. 2. Comparison of the time-response curves of the all-L p e n t a p e p t i d e before and after the alkali-heat t r e a t m e n t . A, all-L (60/~g/ml); t3, all-L (6O / z g / m l ) + washing; C, the t r e a t e d all-L (1. 5 #g/ml); D, the treated all-L (1. 5 /*g/ml) + washing. The a r r o w indicates w a s h i n g b y fresh Ringer solution of skins of B and D groups at 6o min.

Biochim. Biophys. Acta, 115 (1966) 337-344

ALKALI TREATMENT

OF AN ACTIVE F R A G M E N T OF

a-MSH

341

pletely digested by the action of leucine aminopeptidase, the recovery from the treated sample, after leucine aminopeptidase digestion, decreased considerably. The results of L- and D-amino acid oxidase digestions suggested the presence of the racemate in every amino acid component to different extents.

c

c

I'

g C3

control

o

3'o

4

9'o ~o

Time (rain)

Fig. 3. T h e effect of all-D o n frog s k i n p r e v i o u s l y d a r k e n e d w i t h t h e a l k a l i - t r e a t e d all-L p e n t a peptide. - - , all-L (66 # g / m l ) ; • - - O , t h e t r e a t e d all-L (2.2 # g / m l ) ; × . . . . X, c a f f e i n e (I.o m g p e r ml). T h e a r r o w i n d i c a t e s t h e a d d i t i o n of all-D t o t h r e e g r o u p s i n f i n a l c o n c e n t r a t i o n of 5 ° p g / m l . TABLE ANALYSIS

I OF

THE

ALKALI-TREATED

ALL-L

PEPTIDES

Analysis calculation based on glycine recovery.

A m i n o acid

Leucine aminopeptidase digestion* (12moles)

Acid hydrolysis

Acid hydrolysis + L-amino acid oxidase * *

Acid hydrolysis + D-amino acid oxidase * *

Bacterial Micr@iological assay (~moles)

Glycine Histidine Phenylalanine Arginine Ornithine Tryptophan

0.20 0. 51 0.22 o. 15 0.07 0.20

I.O i.o 1.12 o.76 0.24

I.OO 0.22 0.45 o.42 o.21

I.OO 0.81 0.64 o.6I 0-33

-L- : 0.78 ----

.

.

.

.

* F r o m i # m o l e of t h e p e p t i d e . ** A n a l y s i s c a l c u l a t i o n b a s e d o n g l y c i n e r e c o v e r y .

Microbiological assay revealed the racemization of histidine residue to the extent of 22 %. Paper chromatography of the hydrolysate by c~-chymotrypsin or trypsin showed that a considerable amount of the starting material remained undigested. DISCUSSION

When the all-L pentapeptide was submitted to the alkali-heat treatment for lO-6O min, its melanocyte-stimulating activity was always enhanced nearly forty times. Chemical analysis of the resulting peptide showed that, during this treatment Biochim. Biophys. Acta, I 1 5 (1966) 3 3 7 - 3 4 4

342

K. H A N O , M. K O I D A , H. Y A J I M A , K. K U B O , T. O S H I M A

no cleavage occurred in the peptide chain, but one quarter of the arginine was converted to ornithine. The formation of ornithine was identical with the observation made by GESCHWIND AND L13 during the alkali treatment of fi-MSH, and seems responsible for the tailing phenomenon of the treated all-L pentapeptide on paper chromatography. Further studies, after leucine aminopeptidase digestion of the product, and L- and D-amino acid oxidase digestions of the acid hydrolysate, led to the conclusion that four amino acid residues, histidine, phenylalanine, arginine and tryptophan, were racemized to different extents. Paper chromatographic examination of the hydrolysate by c~-chymotrypsin or trypsin showed that the peptide bonds of the alkali-treated peptide had been rendered highly resistant towards the attack of these peptidases. TABLE

II

INDIVIDUAL POTENCY OF THE STEREOISOMERS OF THE ALL-L PENTAPEPTIDE

Peptides

IU/g

Potency of all-L was taken as z.o

L- His--L- PIle--L- A r g - L - T r y - G l y

7" I ° a

I.O

D-His--L- P h e - L - A r g - L - T r y - G l y

o

L- H i s - D - P h e - L - A r g - L - T r y - G l y *

i~

2. lO s

L- HiS--L- P h e - D - A r g - L - T r y - G l y * *

o

L- His--L- P h e - L - A r g - D - T r y - G l y

I - IO ~

D-His-D-Phe-D-Arg-D-Try-Gly

o

Practically inactive 16o Practically inactive I7 o

* R e p o r t e d l l : 3" IO5. ** R e p o r t e d l a : 3 ' IO4"

Alkali-induced racemization of amino acid residues in methionylglutamylhistidyl-phenylalanylarginyltryptophylglycine which is present in c~-MSH, fl-MSH and ACTH was mentioned by previous investigators a-4. Our present results indicate that treatment with alkali indeed causes racemization in every constituent amino acid residue of the all-L pentapeptide. However, as far as melanocyte-stimulating activity i n vitro is concerned, potentiation is the only phenomenon we have observed. This has not been mentioned by other authors 12. Neither retardation nor prolongation effect could be detected in our alkali-treated all-L pentapeptide. We wish to discuss here the possible relationship between the potentiation effect of alkali-treated pentapeptide and its structure in the light of the melanocyte-stimulating activities of our synthetic stereoisomers of the pentapeptide: D-histidine 2a, D-phenylalanine*, D-arginine*, D-tryptophane 23 and all-D ~, ~ analogues shown in Table II. Previously, the potency of a given synthetic peptide was determined by the procedure of SmZUME et al. 26 using natural ~-MSH or extract of pituitary as a standard. It is difficult to compare the potencies of these synthetic peptides in this manner, since their activities are about one millionth of that of ~-MSH. We feel it to be more reasonable to compare the relative potencies of these isomers by using the darkening value (decrease in reflectance) produced by a constant amount of the all-z pentapeptide (33/~g/ml) as a standard. In this assay * Unpublished.

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343

system, the D-histidine analogue was found practically inactive, and the D-tryptophan analogue had 17 times more activity than the all-L pentapeptide. The Darginine analogue has been reported by HOFMAN?; AND LANDE13 to possess a potency equivalent to that of the all-L pentapeptide. But the activity shown by our synthetic peptide has been judged as practically zero. As previously mentioned by SCHNABEL AND LI '1, we also observed that replacement of the phenylalanine residue by the D-isomer results in an increase in its potency, but the observed activity of our synthetic D-phenylalanine analogue was higher than that reported by these authors, the estimated potency being 16o times that of the all-L pentapeptide. These results indicate that in this pentapeptide sequence, the amino acid component which has to be in the L-configuration for the peptide to exhibit natural darkening activity is histidine. Replacement of the phenylalanine or the tryptophan residue by the respective D isomer results in an increase in the potency of the peptide. The observation that certain synthetic pentapeptides with D-amino acid residues exhibit higher activity than the corresponding all-L pentapeptides seems to explain the potentiation phenomenon produced by the alkali-treated pentapeptides which are raeemized. It could be that the potentiation effects of the peptide having this racemized sequence were greater than the decrease in the darkening effect. The observations made during studies on the alkali-treated all-L and the synthetic pentapeptides imply that alkali-treated a-MSH, fl-MSH and ACTH, because of their molecular structure, are capable of potentiating their MSH activities. This could perhaps be extended to explain the phenomenon already known: the survival of the MSH activity in alkali-treated crude pituitary extractS, 6. As mentioned above, the potentiation phenomenon has not been observed in alkali-treated a- and fi-MSH but has been noted in alkali-treated ACTH 1, 4,7, s. For this discrepancy, PICKERING AND LI TM have mentioned that the potentiation effect was observed only in alkali-treated peptides that have an unsubstituted serine residue in the N-terminus as ACTH has. They offered no explanation of the chemical changes responsible for this effect. We suggest as a possible explanation that potentiated MSH activities would be decreased to a greater or lesser extent by some effect of NaOH on the hormone molecules, and such loss of activity could lead to the appearance or disappearance of the potentiation phenomenon in these treated hormones. The prolongation effect produced by alkali-treated hormones in vivo is probably due to racemization of peptide bonds as suggested by GESCI-IWlNDAND LI ~ and LEE AND B.-JANUSCH 2, since the raeemized peptides would be less susceptible to the attack of various proteolytic enzymes. For an explanation of the retardation and prolongation effects of alkali-treated a-MSH 2 in the systems in vitro, further studies are required.

ACKNOWLEDGEMENTS The authors express their sincere appreciation to Dr. I. CIIIBATA of Tanabe Pharm. Co. for the microbiological assay of histidine. They also express their gratitude to Professor S. UYEO of Kyoto University for his encouragement during the course of this investigation.

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K. HANO, M. KOIDA, H. YAJ1MA, K. KUBO, T. 0SHIMA

REFERENCES I T. H. LEE, A. B. LERNER AND V. B.-JANUSCH, Ann. N . Y . Acad. Sci., IOO (1963) 658. J. Biol. Chem,, 238 (I963) 2oi2. 3 I. I. GESC~W~D AND C. H. LI, Arch. Biochem. Biophys., lO6 (i964) eoo. 4 B. T. PmKERINO AND C. H. LI, Arch. Biochem. Biophys., Io 4 (i964) i19. 5 L. T. HOaBEN AND C. GORDON, J. Exptl. Biol., 7 (193 o) 286. 6 F. W. LANDGREBE AND G. M. MITCHELL, Quart. J. Exptl. Physiol., 39 (I954) I1. 7 R. G. SHEPHERD, S. D. WILLSON, I(. S. HOWARD, P. H. bELL, D. S. DAVIES, S. b. DAVIS, E. A. EIGNER AND N. E. SHAKESPEARE, J. Am. Chem. Soc., 78 (I956) 5067. 8 F. L. EN~EL, Vitamins Hormones, I9 (I96I) 189. 9 C. H. LI, Lab. Invest., 8 (1959) 574. lO C. H. LI, E. SCHNABEL AND D, CHLTNG,J. Am. Chem, Soc., 82 (x96o) 2062. i1 E. SCHNA~EL AND C. H. LI, J. Am. Chem. Soc., 82 (196o) 4576. i2 B. T. PICKERING AND C. H. LI, Biochim. Biophys. Acta, 62 (I962) 47513 K. HOFMANN AND S. LANDE, dr. Am. Chem. Soc., 83 (I961) 2286. 14 K. HOF~ANN AND H. YAJIMA, Recent. Progr. Hormone Res., i8 (I962) 4 I. 15 E. A. PETERSON AND I-I. A. SOBER, dr. Am. Chem. Soc., 78 (I956) 75 L I6 ~. M. PARTRIDGE,Biochem. J., 42 (I948) 9_38. 17 J. F. ROLAND AND A. l~'f. GROSS, Anal. Chem., 26 (I954) 502. I8 A. b . LERNER AND M. R. \¥RIGHT, 2Vlelhods Biochem. Analy., 8 (I96o) 295. 19 S. MOORE, D. H. SPACEMAN AND W. H. STEIN, Anal, Chem., 3° (I958) II85. 20 D. H. SPACKMAN, E. L. SMITH AND D. M. /~RO~VN, J. Biol. Chem., 212 (1955) 255. 2i I~. HOFMANN AND H. YAJIMA, J. Am. Chem. Soc., 83 (I96I) 2289. 22 R. J. bLOCK, in P. ALEXANDEr~ AND R. J. bLOCK, A Laboratory Manual of Analytical Methods of Prolein Chemislry, P e r g a m o n Press, London, I96o, Vol. 2, p. 4 I. 23 H. YAJIMA AND I(. t(UBO, Biochim. Biophys. Acta, 97 (1965) 596. 24 K. HANO, ),{. KOIDA, 1~2. KUBO AND H. YAJIMA, Biochim. Biophys. Acta, 90 (I964) 2oi. 25 }J. YAJIMA AND K. KUBO, .J. Am. Chem. Soc., 87 (I965) 2039. 26 K. Smzus~E, A. B. LERNER A~D T. B. FITZPATRICK,Endocrinology, 54 (I954) 553. 2 T . H . LEE AND V. b.-JANUSCI-I,

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