Isolation and characterization of melanocyte stimulating factors from the pituitary gland of the grey mullet, Mugil cephalus

Camp. B&hem.

Physiol., 1974, Vol. 49A, pp. 323 to 329. Pergamon Press. Prkted in Great Britain

ISOLATION AND CHARACTERIZATION OF MELANOCYTE STIMULATING FACTORS FROM THE PITUITARY GLAND OF THE GREY MULLET, MUGIL

CEPHALUS* JOE Department

R. LINTON

and M. S. PROCTOR

of Biology, University of South Florida, Tampa, (Received

21 August

Florida 33620, U.S.A.

1973)

Abstract-l. Three melanocyte stimulating principles were extracted from the pituitary of the grey mullet, Mugil cephalus. 2. The extracts were purified by gel filtration on Sephadex G-25 and by ion exchange chromatography on CM-Sephadex C-25. 3. The specific activities of the substances at all stages of the isolation were determined. 4. The molecular weight of the principles was estimated to be in the lower 2000’s. 5. The activity of mullet melanocyte stimulating hormone was found to be blocked by alpha adrenergic blockade but not by beta blockade. 6. One of the MSH active principles is tentatively identified as ACTH. INTRODUCTION MELANOCYTE stimulating

hormone (MSH) from teleost fishes has received little attention since the initial study done by Burgers (1963) on the cod. Geschwind (1966) compared the positions of two MSH-active fractions from the cod with mammalian MSH on CM-cellulose columns. MSH-active fractions from the pituitary of the carp, Cyprinus carpio, and the salmon, Salmo salar, were reported by Fontaine-Bertrand et al. (1969). Lowe and Pickering (unpublished data cited by Pickering and Heller, 1969) reported MSH activity in the pituitary of a Holocephalan, Hydrolagus collei. Because of the relatively small amount of knowledge on fish MSH, this study was undertaken to isolate and characterize the peptides with MSH activity in the pituitary of the grey mullet, Mugil cephalus. MATERIALS Pituitary

AND METHODS

glands

Pituitary glands were taken from 180 mullet which were captured by gill net in Boca Ciega Bay, Pinellas County, Florida. The glands were placed in dry acetone cooled with dry ice as they were taken from the fish. The cold acetone was changed four times before * This study was partially Grant No. GB-8019.

supported

by National 323

Science

Foundation

Research

324

JOE R. LINTON AND M. S. PROCTOR

the glands were dried under vacuum and stored at - 10°C. The dry glands were ground to a fine powder with a mortar and pestle. The weight of the resulting powder was 110 mg. Bioassay Specific MSH activities were determined by the method of Lerner & Wright (1960). This assay depends upon the MSH-induced decrease in reflected light from isolated frog skin. Small pieces of skin from the back and legs of Ranapipiens were mounted on plastic rings and soaked in four changes of frog Ringer’s for a period of 2 hr to remove endogenous hormone. After this period, the skins were placed in petri dishes containing 50 ml of Ringer’s solution to which the assay material was added. The reflected light was measured by a Photovolt Multiplier photometer (M SOlA), with its photocell mounted on one eyepiece of a binocular microscope. The skins were placed on the stage of the microscope so that the light reflected through the eyepiece and registered on the photometer. The change in reflected light was determined by taking photometer readings before and 1 hr after addition of the assay material. The assay method reported by Burgers (1960, 1961) using isolated skin of the Florida chameleon, Anolis carolinensis, was used to scan chromatography fractions for MSH activity and in the adrenergic blockade experiments. All activity measurements were compared to standard bovine pituitary extracts (1 unit/mg). Data were statistically analyzed according to Holton (1948) to determine activity relative to the bovine standard. Extraction of MSH fractions One hundred mg of the pituitary powder was placed into 1.5 ml of 1 M acetic acid and homogenized with a Potter-Elvehjem homogenizer. This homogenate was then sonified with a Branson S-75 Sonifier for 15 sec. The homogenate was centrifuged at 10,000 g for 30 min, the supernatant was saved and the pellet resuspended, sonified and centrifuged as before. The supernatants were pooled and peptide content determined by the method of Lowry et al. (1951). The MSH specific activity was determined as above. Separation on Sephadex G-25 Two ml of the acetic acid extract were chromatographed on a 0.5 x 75 cm G-25 column with 1 M acetic acid, as developer. The eluent was collected in 2-ml fractions. The fractions were monitored for absorbance at 280 nm and the Lowry et al. peptide content was determined. MSH-active fractions were located by the lizard skin method of Burgers (1960). The fractions with MSH activity were pooled and the specific activity determined with the frog skin assay. These fractions were then dialyzed, lyophilized and stored at - 10°C. Separation on ion exchange chromatography The above lyophilate was dissolved in 4 ml of 0.005 M ammonium acetate (pH 5.9) and chromatographed on a CM-Sephadex C-25 column (1 x 15 cm). The column was developed by adding, stepwise, the following ammonium acetate buffers: 42 ml of 0.005 M, pH 5.9; 42 ml of 0.08 M, pH 5.9; 42 ml of 0.25 M, pH 5.9; 84 ml of 0.5 M, pH 7.0. The eluent was collected in 2-ml fractions which were monitored for absorbance at 280 nm. The fractions were scanned for MSH activity as before and the Lowry peptide content was determined. Homogeneity test Polyacrilamide disc electrophoresis was used to determine homogeneity of the MSHactive fractions from CM-Sephadex (Ornstein & Davis, 1964). Gel concentration was 30%. A Tris-glycine buffer system (pH 8.3) was used. This system stacks at pH 8.9 and runs at pH 9.5. Peptide bands were located by staining with Coomassie blue.

MELANOCYTE

STIMULATING

FACTORS FROM

325

PITUITARY OF GREY MULLET

Molecular weight estimate Molecular weight was estimated from the elution characteristics of the MSH-active fractions from Sephadex G-25 after the method of Anderson & Stoddart (1966).

Adrenergic blockade Large pieces of solutions: Ringer’s blocker or Ringer’s for 2 hr, the skins

skin from the lizard, A. carolinensis, were incubated in one of the following solution, Ringer’s solution with 10e4 M propanalol, a beta adrenergic solution with 10e4 M phentolamine, an alpha blocker. After incubation were tested for a response to MSH.

RESULTS

Acetic acid extraction Extraction

of the pituitary

with increased

specific

MSH

powder activity

25 per cent of the total activity indicates

a preferential

of the original

1).

acid yielded

While

powder,

a preparation

the yield was low, about this higher

specific

activity

extraction.

TABLE I-SUMMARY

OF ISOLATION AND ACTIVITYDRTERMINATIONDATA

Lowry et al. Folin/ peptide (mg)

Preparation

with 1 M acetic (Table

Factor of increase of specific activity relative to Previous step

Original powder

Specific activity (activity/mg)

Total activity (units)

Pituitary powder

109.00 (total weight)

-

1 *oo

0.48

52.32

Acetic acid extract

4.87

6.92

6.92

3.32

16.17

Sephadex G-25 active fraction

1.08

CM-Sephadex Peak 1 Peak 2 Peak 3 Peak 4

0.81 0.56 0.23 0.02 0.02

3.81 -

26.33 -

12.64 -

13.65 -

0.02 0.12 29.36 19.78

0.54 3.04 773.13 520.83

0.26 1.46 371.10 250.00

0.15 0.34 7.42 5.00

Sephadex

G-25 chromatography

Figure 1 shows the elution pattern of the MSH-active fractions plotted against absorbance at 280 nm. The active fractions as measured by the A. carolinensis skin assay were contained in fractions 13-30 ml. The peak showed activity at a dilution of 1 x 10-4. As seen in Table 1, this peak contained 1.08 mg of peptide per ml. The specific activity shows an increase of twenty-six times that of the original powder. Fractions 16-28 ml were pooled for chromatography on C-25 CMSephadex.

326

JOE R. LINTON ANDM. S. PROCTOR -10-S

20 Sephadex

40 C- 25

eluent ,

ml

FIG. 1. Elution pattern of MSH activity from an acetic acid extract of mullet pituitaries on Sephadex G-25. The activity curve as measured by the in V&O color change of A. curoZinensis is superimposed on the absorbance at 280 nm.

Purajicatim on CM-Sephadex C-25 Figure 2 shows the elution pattern of the active fractions from Sephadex G-25 subjected to chromatography on CM-Sephadex C-25. As indicated by the absorption at 280 nm, four distinct peaks emerged from the column. All of these peaks had MSH activity as determined by the lizard skin assay. Peaks 3 and 4 contain high MSH activity and are eluted with 0.25 and 0.50 M ammonium acetate respectively. The fourth peak was not detectable with the Lowry et al. peptide determination and presumably contains less than 0.01 mg of peptide per ml. Peak 1, which did not bind to the column, has low activity and appeared to be heterogeneous.

CM - Sephadex

C-25

eluent, ml

FIG. 2. Elution pattern of MSH-active fractions from Sephadex G-25 column on CM-Sephadex C-25. Absorbance at 280 nm is superimposed on the activity as determined by the Anolis carolinensis assay.

MELANOCYTE

STIMULATING

FACTORS FROM PITUITARY

OF GRRY MULLET

327

Peak 2, which eluted with the 0.08 M buffer, appears to be a discrete peptide with low MSH activity. This could be ACTH because of the similarity in size and reduced MSH activity. Activity determinations The specific activity of all preparations relative to the bovine standard was determined at each step of the isolation procedure. The results of these determinations are summarized in Table 1. The determinations will not be discussed individually except those for peak 4 from the CM-Sephadex column. Since the amount of peptide in this fraction could not be determined because it was less than 0.01 mg per ml, it was not possible to calculate the specific activity of the isolate. It was possible, however, to determine the total activity of the 2-ml fraction showing the greatest absorbance at 280 nm. A small sample of peak 4 was matched with a 2.5 mg/ml solution of the standard. These solutions were diluted serially until the standard was at the usual assay concentration. An assay was then run comparing the activities of the two solutions. The activities were found to be not significantly different. These results indicate that the peak 4 activity is equal to the activity of 2 ml of a 2.5 mg/ml solution of standard, or 5 units of total activity. Since the total activity of 2 ml of peak 4 is 5 units, and the peptide content is less than 0.02 mg, the specific activity can be estimated to be 5/O-02 or over 250 times that of the standard. Homogeneity test When concentrates of the CM-Sephadex peaks were examined for homogeneity by disc gel electrophoresis, peak 1 was found to be heterogeneous as expected, peaks 2 and 3 each showed distinct single bands and peak 4 showed a faint single band when stained with Coomassie blue. Molecular weight estimation Figure 3 shows the results of the estimation of molecular weight from the elution pattern of the MSH active peak from the Sephadex G-25 column by the method reported by Anderson & Stoddart (1966). The fraction with the greatest activity was located in the elution position corresponding to an estimated molecular weight of 2373. Adrenergic blockade Figure 4 shows that pre-incubation of A. carolin& skin with 1 x 10-h M phentolamine HCI, an alpha adrenergic blocking agent, completely blocks the activity of the active fraction from Sephadex G-25. High concentrations of the fraction overrides the alpha blockade. An identical experiment, using the beta blocker, propanalol HCI, showed no effect on MSH activity. These results are similar to those of Goldman & Hadley (1970a, b), who found that the activity of mammalian MSH was subject to aZpha blockade but not to beta blockade.

JOE R. LINTON ANDM. S. PROCTOR

328

Sephodex

FIG. 3. Graphical

estimation

C-

25

eluent,

ml.

of the molecular weight of MSH-active from Sephadex G-25.

peptides

-

0.5

FIG. 4. The effects of the a2pha adrenergic blocking agent, phentolarnine HCl, on the MSH darkening response of A. Caroline&s skin in vitro. Each point represents the mean value of four skins from four different animals. Vertical lines are standard error of the means. DISCUSSION As a result of this study, three discrete principles with MSH activity have been isolated from the pituitary of the grey mullet, M. cephalus. Two of these substances have been identified as true melanocyte stimulating hormones on the basis First, the isolates showed MSH activity of the following three pieces of evidence. at extremely low concentrations when compared to mammalian MSH in the same assay. Second, the estimation of the molecular weight of the factors places them in

MELANOCYTESTIMULATINGFACTORSFROM PITUITARYOF CXEY MULLET

329

the same size range (mol./wt. 1500-2500) as mammalian MSH and Squalus acanthias MSH. Third, the activity of the fractions was blocked by alpha adrenergic blockade in the same way that mammalian MSH activity is blocked. A third principle with MSH activity is tentatively identified as ACTH because of the low specific MSH activity and similarity of size. Burgers (1963) and Geschwind (1966) both found only two MSH active principles in the pituitary of the cod, Gadus morhua. In both cases the factors were identified as true MSH. Fontaine-Bertrand et al. (1969) reported both MSH and ACTH activities in the pituitary of the carp, C. carpio, and the salmon, S. salar. It appears from the few investigations which have been undertaken that there is variation in both the number and kinds of melanocyte stimulating hormones present in the pituitaries of teleost fishes. REFERENCES ANDERSOND. M. W. & STODDARTJ. F. (1966) Some observations on molecular weight estimation by sieve chromatography. Anal. Chem. Acta 34, 40146. BURGERS A. J. C. (1960) Electrophoretic behavior of pituitary melanocyte-stimulating activities of vertebrate origin. 1st Intern. Congr. Endocrinol., Copenhagen. Session IV f. No. 165. Abstract, pp. 329-330. BURGERSA. J. C. (1961) Occurrence of three electrophoretic components with melanocytestimulating activity in extracts of single pituitary glands from ungulates. Endocrinol. 68,698-703. BURGERS A. J. C. (1963) Melanophore-stimulating hormones in vertebrates. Ann. N. Y. Acad. Sci. 100, 669-677. FONTAINE-BERTRANDE., DAVEAU-VAUTIERM. & FONTAINE Y. A. (1969) ActivitCs corticotrope et mClanophorotrope des extraits hypophysaires de deux poissons tClCostCens (carpe et saumon). Dissociation anatomique de ces deux activitCs. J. Physiol. Paris 61, 493-506. GESCHWIND I. I. (1966) Chemistry of the melanocyte-stimulating hormones. In Structure and Control of Melunocytes (Edited by DELLA PORTA G. & MUHLBOCKO.), pp. 28-44. Springer-Verlag, Berlin. GOLDMANJ. M. & HADLEY M. E. (1970a) Evidence for separate receptors for melanophore stimulating hormone and catecholamine, regulation of cyclic AMP in the control of melanophore responses. Br. J. Pharmac. 39, 160-166. GOLDMANJ. M. & HADLEY M. E. (1970b) Cyclic AMP and adrenergic receptors in melanophore responses to methylxanthines. Eur0p.J. Pharmac. 12, 365-370. HOLTON P. (1948) A modification of the method of Dale and Laidlaw for standardization of posterior pituitary extract. Br. J. Pharmac. 3, 328-334. LERNER A. B. & WRIGHT M. R. (1960) I II vitro frog skin assay for agents that darken and lighten melanocytes. In Methods of BiochemicaZ Analysis (Edited by GLUCK D.), Vol. 8, pp. 295-307. Interscience, New York. LOWRY 0. H., ROSEBROUGHN. J., FARR A. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. ORNSTEIN L. & DAVIS B. J. (1964) Disc electrophoresis-I. Background and theory. Ann. N.Y. Acad. Sci. 121, 321-349. PICKERINGB. T. & HELLER H. (1969) Oxytocin as a neurohypophysial hormone in the holocephalian elasmobranch fish, Hydrolagus collei. J. Endocr. 45, 597-606. Key Word Index---Melanocyte phentolamine.

hormone ; pituitary; mullet; Mugil cephalus ; propanalol ;