Purification of the proteinic brain hormone of the silkworm, Bombyx mori

J. Insect Physiol., 1969, Vol. 15, pp. 1981 to 1990. Pergamon Press. Printed in Great Britain

PURIFICATION OF THE PROTEINIC BRAIN HORMONE OF THE SILKWORM, BOMBYX MORI MASAYOSHI

YAMAZAKI

and MASATOSHI

KOBAYASHI

SericulturalExperiment Station, Suginami-ku, Tokyo, Japan (Received 27 January

1969)

Abstract-The proteinic brain hormone (the prothoracotropichormone) was purifiedfrom the extractof brainsof Bombyx pupae by means of chromatography on columns of DEAE- and CM-cellulose and gel filtrationon Sephadex G-100. The purified brain hormone was active at the 0.02 pg level causing adult development in the Bow&x assay. Overall purificationwas about 2000-fold. The isoelectric point of the hormone was determined to be 8.35 to 8.65 by isoelectric focusing. The hormone was inactivatedby treatment with trypsin, pronase, or nagarse, but was not inactivatedby chymotrypsin or sialidase. Its molecularweight was estimatedby gel filtrationon Sephadex G-100 to be about 20,000. A highly purified preparation was made by means of zone electrophoresis on acrylamidegel. The hormone may be a glycoprotein. INTRODUCTION

A CRUDEhormone extract from insect brains was first noted by KOBAYASHIand KIRIMURA (1958). KIRIMURA et al. (1962) reported that the active substance finally obtained from the brain in the silkworm was identical with cholesterol. Subsequently, they found an additional active factor (KOBAYASHI et al., 1962; KOBAYASHI, 1963). The latter, on further investigation, appeared to be of a proteinic nature (KOBAYASHIand YAIMAZAKI,1966), which was in accord with the finding of ICHIKAWAand ISHIZAKI(1961, 1963). The latter purified the hormone by means of gel filtration and chromatography on a cellulose-ion exchanger (ISHIZAKIand ICHIKAWA,1967). MATERIALS

AND

METHODS

Source of brain hormone extract Bombyx

brains were extirpated from pupae and stored at - 20°C.

Bioassay for brain hormone activity The dauer pupae of Bombyx mori were used as test objects for the bioassay as previously described (KOBAYASHIand YAMAZAKI, 1966). DEAE-cellulose DEAE-cellulose (O-65 m-equiv/g) was a product of Serva Co. Ltd. Before use the fine particles were removed by decantation after washing with deionized 103

1981

1982

MA~AYOSHI YAMAZAKI AND MASATOSHI KOBAYASHI

water. The washed DEAE-cellulose was transferred to a Biichner funnel and converted to the OH form by passing O-4 N NaOH through it. It was washed with deionized water until the filtrate was neutralized, and was then treated with 0.01 M Tris-HCl buffer at pH 8.0. CM-cellulose CM-cellulose (O-83 m-equivjg) was a product of Serva Co. Ltd. After CMcellulose was converted to the H form by passing 0.4 N HCl through it, it was treated with O-01 M phosphate buffer at pH 7.5 or 7~0. Sephadex

G-100

Sephadex G-100 was a product of Pharmacia 0.05 M phosphate buffer at pH 6.8. Proteins as standard

Co. Ltd.

It was washed with

sample

Yeast cytochrome-c was a gift of the Sankyo Co. Ltd. Crystalline chymotrypsin was obtained from the Mochida Pharm. Co. and pepsin from the Worthington Biochemical Corporation. Determination

of protein concentration

The protein concentration of column effluents was determined from absorbancy at 280 rnp. Cytochrome-c was recorded at 550 rnp. The protein concentration of each fraction used for the bioassay was determined by Lowry’s method using crystalline egg albumin as a standard (LO~NRY et al., 1951). All the purification procedures were performed in a cold room at 4°C. RESULTS

Purification

of proteitiic

AND

DISCUSSION

brain hormone

Extraction of the crude extract from Bombyx brains. Acetone-dried brains from about 100,000 pupae were homogenized in 120 ml of O-1 M phosphate buffer at pH 6.8 with a glass homogenizer; the supematant was obtained by centrifuging the homogenates at 5000 revfmin for 15 min. This procedure was repeated three times. The final volume was about 350 ml. Chromatography on the DEAE-cellulose column. About 350 ml of supematant was dialysed overnight against 0.05 M Tris-HCI buffer at pH 8-O; the dialysed solution was diluted with 2 ~01s. of deionized water and placed on a column of DEAE-cellulose (3.5 x 30 cm) equilibrated with the same buffer. The column was washed with the buffer and then eluted with the buffer containing O-1 M sodium chloride. The two eluates were combined and dialysed against deionized water and then lyophilized (weight = Z-1123 g). Chromatography on the CM-cellulose column. About 2 g of the afore-mentioned active material was dissolved in 100 ml of 0.01 M phosphate buffer at pH 7.5 ; the solution was dialysed against the same buffer overnight and placed on a column of CM-cellulose (3.5 x 30 cm) and equilibrated with the buffer. As shown in Fig. 1,

PURIFICATION OF PROTBINICBRAINHORMONEOF SILKWORM

1983

after the column was washed with the buffer and until the appearance of peak II, the active material was eluted with the buffer containing 1 M sodium chloride at a flow rate of 30 ml/lx. The effluent was collected in 3 ml fractions. The fractions in each peak were pooled and dialysed against deionized water and lyopbilized. Weights of peaks I, II, and III were l-369,0-273, and O-085 g respectively. Positive results were found in peaks I and III and the specific activity on the bioassay was much higher in peak III than in peak I.

N

lw

.

I.

I.

-I

P- III

I

P-II

TUBE NUMBER

FIG. 1. Chromatography of the brain hormone fraction DEAE-I on a column of CM-cellulose. The black band shows the range of brain hormone activity.

50

100

TUBE NUMBER

FIG. 2. Chromatography of the brain hormone fraction P-III on a column of CM-cellulose. The black band shows the range of brain hormone activity.

1984

MASAYOSHI YAMAZAKI AND MASATOSHI KOBAYASHI

Second chromatography on the CM-cellulose column. The active fraction (peak III) obtained by the first chromatography was dissolved in 2 ml of 0.01 M phosphate buffer at pH 7.5 and dialysed against the same buffer overnight and placed on a column of CM-cellulose (1.5 x 30 cm) equilibrated with the buffer. The column was eluted with the buffer containing a linearly increasing concentration of sodium chloride at a flow rate of 20 ml/hr. The effluent was collected in 5 ml fractions. A typical chromatographic pattern is shown in Fig. 2. After elution with 100 ml of the starting buffer (O-01 M phosphate buffer, pH 7.5) a reservoir containing the second buffer (O-01 M phosphate buffer containing O-5 M sodium chloride, 300 ml, pH 7.5) was connected to a mixing chamber filled with 300 ml of the starting buffer.

TUBE NUMBER FIG. 3. Re-chromatography

CM-cellulose.

of the brain hormone fraction in Fig. 2 on a column of The black baud shows the range of brain hormone activity.

Every second fraction was bioassayed. Positive results were found in fractions 36 to 58. For further purification, the active fractions in the foregoing bioassay were pooled and re-chromatographed on a column of CM-cellulose (1.5 x 30 cm). The conditions of elution were the same as those used in the second chromatography with the exception that the starting buffer (0.01 M phosphate at pH 7.0) and the second buffer (0.01 M phosphate buffer at pH 7-O containing O-4 M sodium chloride) were used. The effluent was collected in 3 ml fractions (Fig. 3). Every second fraction was assayed. Fractions 74-94 showed activity. These fractions were pooled and lyophilized in three groups, namely 65 to 75, 76 to 83, and 84 to 95. GeZJiZtration on Sephadex G-100. Each of the lyophilized fractions (F-I, II, and III) was dissolved in 1 ml of 0.05 M phosphate buffer at pH 6.8 and placed on a column (1 x 50 cm) of Sephadex G-100 equilibrated with the same buffer. The column was eluted with the buffer at a flow rate of 10 ml/hr. The eflluent was collected in 1.8 ml fractions.

PURIFICATION OF PROTRINIC BRAIN HORMONE

1985

OF SILKWORM

Every second tube was assayed. As shown in Fig. 4, fraction G-I from F-I was inactive and tubes 17 to 27 of fraction G-III from F-III active, while, although not indicated in Fig. 4, fraction G-II from F-II showed positive bioassay although this fraction was mixed with the most active fraction G-III. '4x103

.iI*

. I\ . I.

G-111

G-l

.O’

.

.-.#.I

10

1 20

‘\...‘‘-,_ 30

g E

4 I’

I ._,_+I{ 1 !\ I

-2x103 4 ‘;r’ e 8 z

‘I I- ‘.C*“\-

10

2(

TUBE NUMBER

FIG. 4.

Gel filtration of the fraction F-I and F-III in Fig, 3 on a column of Sephadex G-100. The black columns show brain hormone acitvity. One unit of brain hormone activity is defined as the amount of brain hormone that is necessaryfor emergence of a dauer pupa.

Chromatography on the DEAE-cellulose column. The active subfractions were combined and placed on a column of DEAE-cellulose (1 x 30 cm) equilibrated with Tris-HCl buffer at pH 9.0. The column was first eluted with the buffer and then with a gradient prepared in a mixing chamber containing 100 ml of O-01 M Tris-HCI buffer at 9-O and a reservoir containing 100 ml of the buffer plus 0.3 M sodium chloride at pH 9.0. The flow rate was 15 ml/hr. The effluent was collected in 1.8 ml fractions. Two fractions were separated as shown in Fig. 5. Positive bioassay was obtained for fraction D-II. Results of all these procedures are summarized in Table 1. One Bombyx unit of hormone corresponds to 0.02 pg of the purified material. Homogeneity and characteristics of the purified brain hormone Electrophoresis on acrylamide gel. Electrophoresis of the purified hormone on acrylamide gel was carried out following the method of KOBAYASHI and YAMAZAKI (1966). The electrophoretic patterns showed two bands as shown in Fig. 6. Action of proteolytic enxymes on the brain hormone. Action of proteolytic enzymes on the purified hormone was investigated. To this end, five solutions were prepared, each containing 4 pg of the purified hormone in 1 ml of 0.1 M phosphate buffer at pH 8 -0 (in the case of sialidase 0.02 M citric acid-sodium phosphate, pH 6-O). Each solution received 4 pg of one of the following enzymes: trypsin, chymotrypsin, nagarse, pronase, or sialidase. After incubation at 30°C for 1 to 24 hr, O-01 ml of each solution was injected into each assay animal. The results are

MASAYOSHI YAMAZAKI AND MA~ATOSHI KOBAYASHI

1986

TABLE ~-SUMMARY

Fractions Crude extract DEAE-cellulose chromatography CM-cellulose chromatography (P-I) CM-cellulose chromatography (P-III) Gel filtration on Sephadex G-100 (G-III) DEAE-cellulose chromatography (D-II)

OF PURIFICATION OF THE BRAIN HORMONE

Total protein (mg)

Total brain hormone units

Specific activity (units/mg)

Recovery in each step (%)

57,025 2112

124,500 106,000

22 50

100 93.2

1369

27,380

20

22.2

42,600

500

34.2

0.55

27,360

45,454

22.2

0.45

20,450

45,454

16.4

85.2

D-II

!l’UBE’ NU&R

FIG.

5.

Chromatography of the fraction G-III in Fig. 4 on a column DEAE-cellulose. The black band shows brain hormone activity.

of

summarized in Table 2. Although not indicated in the table, control studies were carried out simultaneously with the bioassays. It appears that the purified hormone is inactivated by trypsin, pronase, and nagarse. The fact that the crude extract is not inactivated by trypsin (KOBAYASHI and YAMAZAKI, 1966) may suggest that the crude extract contains some sort of trypsin inhibitor. Effects of some other treatments. One ml of aqueous hormone solution (4 pg/ml) was treated at 100°C for 15, 30, or 60 min; 0.01 ml of the treated solution was used in each bioassay. Four r_Lgof the purified hormone was dissolved in 1 ml of O-1 M sodium carbonate, pH 10.0, or 10 N HCI, pH 1.4. Incubation was for 24 or 48 hr at 25°C; 0.01 ml of solution was then used in each bioassay. The results obtained are presented in Table 3. Clearly, the brain hormone was very stable at boiling point, acid or base. Molecular weight of the brain hormone as estimated by gel$ltration with Sephadex G-100. Sephadex gel filtration was used following the method of several workers

t li -

origin

I ÷

|~;l~_,ctrophoretic pattern of purified br~lin h~rmont, on ~l~'r~,'lamidt~~ l .

PURIFICATIONOF PROTEINICBRAIN HORMONEOF SILKWORM

1987

TABLE 2--INACTIVATION OF PURIFIEDBRAIN HORMONEBY PROTBOLYTICBNZYMBS Enzymes used for inactivation (digestion time hr)

No. of dauer pupae

No. of moths emerging

No. of pupae dead

Trypsin

(1) (2) (4) (12)

6 6 6 6

2* 1* 0* 1*

0 0 0 0

Chymotrypsin

(1) (2) (4) (24)

6 6 6 10

5 6 5 5

1 0 0 2

(4)

6 6 6

2* 1* 2*

0 0 0

Nagarse

(1) (2) (4)

6 6 6

0” 2” 1*

0 0 0

Sialidase

(1)

6

6

:i;

6

3 5

Pronase

Results obtained 4 weeks following injection at 25°C. * Inactivated.

TABLE 3-TRBATMIZNT OF PURIFIEDBRAIN HORMONB

PH

10

1.4

No. of dauer pupae

No. of moths emerging

No. of pupae dead

15 mill* 30 min* 1 hr*

6 6 6

5 6 3

1 0 3

24 hr 48 hr

6 6

6 4

0 1

24 hr 48 hr

6 6

3 3

1

Time

Results obtained 4 weeks following injection at 25%. * Experiments carried out at 100°C.

1

1988

~UASAYOSHI YAMAZAKI and MASATOSHI KOBAYA~HI

(ANDREWS, 1964,1965;SIEGELand MONTY, 1966). One mg of standard proteins (pepsin, chymotrypsin, or yeast cytochrome-c) was dissolved in 1 ml of 0.05 M phosphate buffer at pH 68 and each solution was applied to a column of Sephadex G-100 (1 x 50 cm) equilibrated with the same buffer. The column was eluted with the buffer at a flow rate of 10 ml/hr. The effluent was collected in 18 ml fractions. The standard proteins and the purified ingredient were run separately as shown in Fig. 7. The molecular weight of the brain hormone was estimated to be about 20,000 by comparison with the standard proteins.

lUBE NUMBER

FIG, 7. Gel filtration of purified brain hormone and several proteins of known molecular weight on a column of Sephadex G-100. P: Pepsin; C: chymotrypsin; Y: yeast cytochrome-c; B : purified brain hormone.

Other characteristics of the brain hormone. Recently, isoelectric focusing has proved to be a good method for the determination of isoelectric points (~1) of proteins (CARLSTROMand VESTERBERG, 1967; VESTERBERG et al., 1967). The carrier ampholytes used in this investigation were selected to give a pH gradient between 3 and 10. ,The total carrier ampholytes were 1 g in each experiment. The focusing was carried out at 4°C in a special vertical electrolysis column of 110 ml capacity, equipped with a cooling jacket (LKB Co.). The density gradient was made up of sucrose (O-SO% w/v). C oncentrated phosphoric acid (0.5 ml) was added at the anode to prevent oxidation of the carrier ampholytes and ethylenediamine (0.5 ml) was added at the cathode to prevent reduction. About 10 ,ug of the purified hormone was used in each experiment. After focusing for 48 hr with a final potential of about 700 V, the carrier ampholytes of the column were collected in 2 ml fractions, after which the pH of each fraction was measured at 25°C. For each bioassay 0.01 ml of each fraction was used. Fractions 41 (pH 8.35) to 43 (pH 8.65) showed activity. This result indicates that the isoelectric point of the hormone is in the range of pH 8.35 to 8.65 at 25°C. Considering the importance of glycoprotein in some neurohormones of vertebrates, the sugar content of the purified hormone was determined by the phenolsulphuric acid method (DUBOISet al., 1956).Our most active preparation contained

PURIFICATION OF PROTEINICBRAINHORMONEOF SILKWORM

about 15 per cent glucose equivalent. be a glycoprotein or mucoprotein.

1989

This suggests that the brain hormone may

P" 10 9

a 7 6 5 4 3

1'0

30

50

TUBE NUMBER at 25°C of the FIG. 8. Brain hormone activity (black band) and pH (e-e) fractions taken from a 110 ml column after isoelectricfocusing.

Furthermore, 1 ml of a mixture of chloroform and amyl alcohol (4 : 1) was added to an equal volume of the solution containing the purified hormone (4 pg) and the mixture was vigorously agitated at room temperature on a vortex mixer. The aqueous hypophase was sucked off and subjected three times to the same treatment, and the aqueous phase was then assayed. A negative was injected into test pupae. ISHIZAKI and ICHIKAWA (1967) reported that the brain hormone shows heterogeneous molecular weights, the major components ranging from 9000 to 31,000. In their work the brain hormone was adsorbed on DUE-cellulose (pH 7.8) but not on CM-cellulose. They pointed out that their result differed from ours, and that the difference was due to methodology. However, in our experiment abou.t 22 per cent of the total units of the brain hormone in the starting material was not adsorbed by CM-cellulose but was adsorbed by DEAE-cellulose (KOBAYASHI and YAMAZAKI, 1966). Consequently, the brain hormone purified by ISHIZAKI and ICHIKAWA (1967) probably corresponds to our unadsorbed fraction. Clearly, this is a different molecule from the hormone purified in the present study. GERSCH and ST~~IUWICHNUNG (1968) reported that aqueous extracts of the Periplaneta americana, gave two components upon gel filtration on Sephadex G-75, and that the brain hormone seemed to be proteinic. Recently ,WILLIAMS (1967) reported that the brain hormone might be a mucopolysaccharide on the basis of its heat-stability, its lack of specific absorption at

brains of the cockroach,

1990

MA~AYOSHIYAM~AKI AND MASATOSHIKOBAYASHI

280 m,u, and its insensitivity to classical methods which have been used to remove the last traces of proteins. From these facts we believe that the brain hormone in insects is a protein and consists of several kinds of proteins or polypeptides. Acknowledgements-The authors are deeply indebted to Dr. T. YOKOYAMAof the Silk Science Research Institute for his encouragement and interest extended during the course of this work, and to Professor C. M. WILLIAMS of Harvard University for his invaluable suggestions and for reading the manuscript. The authors thank Dr. K. HIROTA of the Sankyo Co. Ltd. for kindly supplying the yeast cytochrome-c and Dr. T. GAMO of the Sericultural Experiment Station for technical help. REFERENCES ANDREWSP. (1964) Estimation of the molecular weights of proteins by Sephadex gel filtration. Biochem. J, 91, 222-233. ANDRaYs P. (1965) The gel filtration behavior of proteins related to their molecular weight over a wide range. Bi0chem.J. 96, 595-606. CARLSTROMA. and VESTERBERG 0. (1967) Isoelectric focusing and separation of the subcomponents of lactoperoxidase. Acta them. Stand. 21, 271-278. Duso~s M., GILLIE~ K. A., HAMILTONJ. K., REFERSP. A., and SMITH F. (1956) Colorimetric method for determination of sugars and related substances. Analyt. Chem. 28, 350-356. GERSCHM. and S~~EICHNUNG J. (1968) Weitere Untersuchungen zur Kennzeichnung des Aktivationshormones der Insektenhautung. 2. Insect Physiol. 14, 87-96. ICHIKAWA M. and ISHIZAKI H. (1961) Brain hormone of the silkworm, Bombyx mori. Nature, Lond. 191, 933-934. ICHIKAWAM. and ISHIZAKIH. (1963) P ro tein nature of the brain hormone of insects. Nature, Lond. 198, 308-309. ISHIZAKIH. and ICHIKAWAM. (1967) Purification of the brain hormone of the silkworm, Bombyx mori. Biol. Bull., Woods Hole 133, 355-368. KIRIMURAJ., SALTO M., and KOBAYA~HIM. (1962) Steroid hormone in an insect, Bombyx mori. Nature, Lond. 195, 729-730. KOBAVASHIM. (1963) The chemistry and physiology of the brain hormone. Proc. 16th int. Cong. 2001. 4, 226-233. KOBAYA~HIM. and KIRIMURA J. (1958) The brain hormone in the silkworm, Bombyx mori L. Nature, Lond. 181, 1217. KOBAYA~HIM., KIRIMURAJ., and SAITOM. (1962) The brain hormone in an insect, Bombyx mori L. (Lepidoptera). Mushi, 36, 85-92. KOBAYASHIM. and YAMAZAKIM. (1966) The proteinic brain hormone in an insect, Bombyx mori L. (Lepidoptera: Bombycidae). AppE. Ent. Zool. 1, 53-60. LOWRY O., RO~EBROUGH N. J., FARRA. L., and RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. j? biol. Chem. 193,265-275. SIEGELL. M. and MONTY K. J. (1966) Determination of molecular weights and frictional ratios of proteins in impure systems by use.of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductase. Biochim. biophys. Acta 112, 346-362. VESTERF~ERG O., WADSTROMT., VESTERBERG K., SVENSSON H., and MALMGREZNB. (1967) Studies on extracellular proteins from Staphylococcus aureus-I. Separation and characterization of enzymes and toxins by isoelectric focusing. Biochim. biophys. Acta 133, 43545. WILLIAMS C. M. (1967) The present status of the brain hormone. In Insect Physiology (Ed. by BEAMENT J. W. L. and TREHERNE J. E.), pp. 133-139. Oliver & Boyd, Edinburgh.