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Page 1: Separation and purification of distal retinal pigment hormone and red pigment concentrating hormone of the crustacean eyestalk

3 ° 0 SHORT C()MMUNICATIONN

(GB 4465) and the Pennsylvania Department of Agriculture. The expert technical assistance of Mrs. 3IA~v REaLE is gratefully acknowledged.

King Ranch Laboratory of Re2/)roduclive Ph3,siolog3, , School of Veterinary Medicine, Universi O, of Pennsvh,ania, Philadelphia, Pa. (U.S.A.)

I{ALPH L. I'IRIXSTER

i 1¢. L. iSRINSTER, .]. Expll. Zool., I58 (I905) 59. 2 R. L. BRXNSrER, Biochim. Biophys. Acta, ro (r965) 439. 3 [{. L. BRINSTER, J. Repro& Fert., ro (I9f~5) 227 . 4 B. MINIZ, Science, 138 (t962) 594. 5 C. LUT\VAK-I~{ANN, .\:atl.c~rc, 193 (I9()2) 1')53. () l(. J. BLANDAU, in W. ('. h'OUNG, S(?r and Internal Secretions, Vol. 2, Williams and Wilkens,

Baltimore, Md., 3rd cal., [()~)1, p. 797.

Received June 22nd, z9~ 7

Biochi***. l¢iophys. Acta, I.t<'; (i 0~)7) 20g 3oo

BBA 23360

Separation and purification of distal retinal pigment hormone and red pigment concentrating hormone of the crustacean eyestalk

Among the numerous investigations concerning tile neurosecretory eyestalk hormones of crustaceans (for literature see refs, I-3), very few have been directed towards their isolation and purification. A study was therefore started in this labora- tory to purify the different hormones from the eyestalks of PandaIus borealis ~. The investigation resulted in the isolation of a distal retinal pigment hormone, which regulates the movement of the distal retinal pigments, and a red pigment concen- trat ing hormone, which controls tile movements of the erythrophore pigments. Because the original procedure used for their isolation was accompanied by a great loss of both horinone activities a, the isolation procedure was reinvestigated and modified to allow purification of the two hormones in high yields. The present report describes the details of this purification procedure.

Eyestalks removed from living Pandalus borealis, immediately frozen and sub- sequently lyophilized, served as the source of the hormones. Living Palaemon ad@erszls were used as test animals for the bioassays of both hormone activities". The methods were essentially based on procedures already described >-9.

Powdered lyophilized eyestalks (5 g) were wet with distilled water U5 o nil), heated in a boiling-water bath for 4 rain under continuous stirring, and then chilled under running tap water. All further operations were performed in a cold room at 4 .

The insoluble material was centrifuged off (27ooo ~ g for 15 min) and washed. The combined supernatants (I68 ml) were subsequently lyophilized (Fraction i, Table I). The brown powder was repeatedly extracted with distilled water and the solution (6. 5 ml) was adjusted from pH 8.2 to 3.7. A brown precipitate which formed during the pH adjustment was removed by centrifugation (as above) after 2 h of standing, and washed.

]Hochim. Bioph3,s. Acta, t48 (1907) 300-303

Page 2: Separation and purification of distal retinal pigment hormone and red pigment concentrating hormone of the crustacean eyestalk

SHORT COMMUNICATIONS 301

T A B L E I

I S O L A T I O N O F D I S T A L R E T I N A L P I G M E N T H O R M O N E A N D R E D P I G M E N T C O N C E N T R A T I N G H O R M O N E

F R O M E Y E S T A L K S OF P. borealis

Figures are given for 5 g of lyophilized eyestalks.

Fraction Dry weight Total activity (%) Specific activity (rag) (related to weight*)

Distal retinal Red pigment Distal retinal Red pigment pigment concentrating pigment concentrating hormone hormone hormone hormone

I Aqueous extract 1572 IOO ioo I.OO

2 Pooled fractions from Dowex 5 ° W-X2 49.4 44 63 14.2

3 Pooled fractions from DEAE-Sephadex A-25 (a)2i. 3 42 31.5

(b) o,99 61

I .OO

20.2

975

* Arbi t rary unit. Aqueous extract taken as I.OO.

The supernatants were combined (7.4 ml) and applied to a 2.5 cm × 47 cm column of Dowex 50 W-X2 (Kabi, Stockholm, lOO-2OO mesh), prepared in o.I M tri- ethylamine formate buffer and equilibrated to pH 3.7. The chromatography was carried out by a three-step elution system, including o.I M triethylamine formate buffer (pH 3-7) ; o.I M triethylamine acetate buffer (pH 5.5), and o.I M triethylamine solution (pH 11.4). The elution rate was 86 ml/h. Fractions collected for each 15 min were analyzed for absorbance at 280 m~ and for hormone activities. A representative elution pattern is given in Fig. i. The distal retinal pigment hormone activity was

<

1.0

0.5 pH 3.7 ~ l ~ p14 5.5 ~ l ~

1000 2000 3000

EFFLUENT (mL)

pH 11£ 10

- ~ o 4000

Fig. I. Chromatography of distal retinal pigment hormone ( . . . . . ) and red pigment concentrat ing hormone ( . . . . . . . ) on Dowex 5 ° W-X2. A280, ( ). Exper imenta l details are given in the text.

Biochim. Biophys. Acta, 148 (i967) 300-303

Page 3: Separation and purification of distal retinal pigment hormone and red pigment concentrating hormone of the crustacean eyestalk

302 SHORT ( ' ( ) M M U N I ( ' A T I t ) N S

always obtained in a single and sharp peak imnlediately after tile pH change in the: effluent. The red pigment concentrating hormone activity, in contrast, was eluted rather slowly and appeared as a broken band different in shape in each experiment. This variation of the shape of the red pigment concentrating hornmne peak resulted from a simplified bioassay procedure used when assaying the set)arate fractions and did not indicate different components. The simplified procedure, required by the large number of fractions to be analyzed, included injection of only ~me dose f,), em:'h fraction into each of 3 test animals, and therefore the effluent curves show ~mlv tht~ averaged responses for each fraction.

The fractions containing the activities were pooled and subsequently h'(,- philized (Fraction 2, Table I). The brown-yellow residue was dissolved in a small \'(d. of distilled water, and the clear yellow solution (3.5 ml) was adjusted from pH 4.(} to 2.5. The solution was applied to a 1.2 cm lO2 cm column of DEAE-Sephadex A 25 (Pharmacia, Uppsala, nledium), prepared according to the manufacturer and equili- brated to pH 2.5 with o.I M formic acid. The column was eluted at a constant fl(~w rate of 6 ml/h with o.I .XI formic acid, pH 2.5. Fractions eollected for each I5 luin \vl:,rc analyzed for absorbance at 28o m/, and for hormone activities. Although sinKle and completely separated peaks were always obtained for the two activities, there wa~ n(, peak of absorbance at 28o ,n/, corresponding to the red pigment concentrating hormone activity (@ ref. 4)- The fractions containing the two activities were p,~led separately and then subsequently lyophilized (Fractions 3a and b, Table I).

Although the present procedure isolated the two llormones in good yield~, a considerable loss of distal retinal pigment hormone activity was always observed after the chronlatography on Dowex 5 o. Because no (:omparable loss (ff activity was observed for the red pigment concentrating hormone and no distal retinal pignlent hormone activity could be detected elsewhere in the column effluent, this l<)ss ,)f distal retinal pignlent hormone activity needs further investigati,m.

In spite of a considerable increase in their specific activity, ab<mt IOO times f()r the distal retinal pigment hormone and 3000 times fl~r the red pigment c~mc~*ntrating hormone over that of lyophilized eyestalks, it is evident that they still contain im- purities and therefore require further purification. "I'(~ be favourable this must, h,~\v- ever, involve procedures capable ~)f handling large anlounts of starting material, because of the very minute amounts of hormones present in the tissues of the prawn.

No observation was made justifying previous assumptions (@ refs. i 3) that different hornlones are affecting the eoncentration (~f the pigments in the' small and large erythr()phores.

Inactivation bv proteolytic enzymes has previously been ret),~rted for the t~o hormone activities and has been the main argument for a proposed peptide structure of the hormones 8,10. Samples of the two isolated and purified hormones were therefl~re tested in respect to their stability towards hydrolysis with pepsin, trypsin and chymotrypsin (2 times crystallized enzymes, optimal pH). The distal retinal piginent hormone (I mg/ml) proved to be very sensitive to all three enzymes. A loss ,ff more than half of the activity was obtained after treatnlent for 3 ° min at 25 ' , when the enzymes were present in concentrations varying between o.5 to 5 o izg/nll. The re(1 pigment concentrating hormone (5 o /,g/nll) was also rapidly inactivated bv pepsin and chymotrypsin under the same conditions but showed strong resistance to trypsin (5 /~g/ml) which caused no inactivation during 6 h at 25°.

Biochim. Biophys. dcla, I48 (I907) 300-3o3

Page 4: Separation and purification of distal retinal pigment hormone and red pigment concentrating hormone of the crustacean eyestalk

SHORT COMMUNICATIONS 303

These results thus lend further support to the suggestion tha t the two hormones consist of peptides al though a final conclusion must await a chemical invest igat ion

of pure samples. The s tabi l i ty of the hormone preparat ions to acid and alkaline hydrolysis at 25 °

was also tested. The distal ret inal p igment hormone (0.5 mg/ml) was found to be rather sensitive to acid hydrolysis and lost about half of its ac t iv i ty in I M HC1 during 24 h, while the red pigment concentra t ing hormone (o.i mg/ml) was stable under these conditions. In alkaline solution both hormones were rapidly inact ivated. The distal ret inal p igment hormone lost all of its ac t iv i ty wi thin I h in I M NaOH, and under the same condit ions more than half of the red pigment concentra t ing hormone ac t iv i ty was lost.

Thanks are due to Dr. B. SWEDMARK for providing oppor tun i ty to carry out part of this invest igat ion at the Krist ineberg Zoological Station, and to Miss. V. A. ANDERSSON and Miss L. NILSSON for their excellent technical assistance. The in- vest igat ion was aided by grants from the Swedish Medical Research Council (Project No. I3X-I7) and from the Univers i ty of Lund.

Delbartment of Physiological Chemistry, University of Lund, Lurid (Sweden)

LARS JOSEFSSON

i H. CHARNIAUX-COTTON AND L. H. KLEINHOLZ, in G. PINcUS, K. V. THIMANN AND E. B. ASTWOOD, The Hormones, Vol. 4, Academic Press, New York, 1964, p. 135.

2 E. J. W. BARRINGTON, in G. PINCUS, K. V. THIMANN AND E. B. ASTWOOD, The Hormones, Vol. 4, Academic Press, New York, 1964, p. 299.

3 M. FINGERMAN, Physiol. Rev., 45 (1965) 296. 4 L. JOSEFSSON AND L. H. KLEINHOLZ, Nature, 2Ol (1964) 3Ol. 5 L. H. KLEINHOLZ AND F. KIMBALL, Gen. Comp. Endocrinol., 5 (1965) 336. 6 P. FERNLUND AND L. JOSEFSSON, to be published. 7 M. I. SANDEEN AND F. A. BROWN, Physiol. Zool., 25 (1952) 222. 8 L. H. KLEINHOLZ, H. ESPER, C. JOHNSON AND F. KIMBALL, Biol. Bull., 123 (1962) 317. 9 M. I. SANDEEN, Physiol. Zool., 23 (195o) 337.

IO P. EDMAN, l~. F~.NGE AND E. (~STLUND, Proc. 2nd Intern. Syrup. Neurosecretion, Lund r957, Springer-Verlag, Berlin 1958, p. 119.

Received May I2th, 1967

Biochim. Biophys. Acta, 148 (1967) 300-303