Comp. Biochem. Physiol., 1976, Vol. 53II, pp. 133 to 137. Pergamon Press. Printed in Great Britain
ISOLATION AND CHARACTERIZATION OF PROLACTIN FROM THE GREY MULLET, M U G I L CEPHALUS J. T. WOOSLEY AND J. R. LrNTON Department of Biology, University of South Florida, Tampa, FL 33620, U.S.A.
(Received 21 May 1974) Abstract--1. A prolactin-like hormone has been isolated from the pituitary of the grey mullet, Mugil cephalus (Linnaeus), by ultrafiltration and analytical chromatography on G-75 Sephadex. 2. The hormone possessed biological activity in the Gillichthys xanthophore dispersion assay (Sage & Bern, 1971). 3. A sensitive radioimmunoassay for mullet prolactin was developed and validated by comparison with biological activity data. Some immunological similarity between the ovine prolactin molecule and the mullet prolactin molecule was demonstrated by this radioimmunoassay. 4. A molecular weight of 22,400 was indicated by the elution behavior of mullet prolactin from G-75 Sephadex.
INTRODUCTION
THERE have been several attempts to isolate fish prolactin. Chadwick (1965), applied Sephadex chromatography and starch gel electrophoresis to pituitary extracts of several teleosts and elasmobranch species. Activity was measured by the mammary gland and pigeon crop sac bioassays. Little activity was found and no attempt was made to characterize the fish hormone. Pickford et al. (1965) extracted carp pituitaries using a modification of the Bates & Riddle (1935) procedure. The isolated material did not have biological activity as measured by its ability to promote survival of hypophysectomized Fundulus heteroclitis in fresh water. Material from carp and pollack pituitaries has been found to cross react with ovine and bovine prolactin antibodies (Emmart et al., 1966, Emmart & Wilhelmi, 1968). This finding was exploited in an attempt to isolate fish prolactin by Emmart & Bates (1968). Anti-ovine prolactin antibodies were added to fractions from carp and pollack pituitaries. The precipitated antigen-antibody complex was collected, dissolved, and chromatographed on G-200 sephadex. Several broad peaks were obtained which reacted immunologically with antiovine antibodies. These results indicate that fish prolactin may form aggregates at low pH as does the mammalian hormone (Andrews 1966; Cole & Li, 1959; Ferguson & Wallace, 1960; Riesfeld et al., 1964; Squire et al., 1963). The material isolated in this manner did not have biological activity using the survival of F. heteroclitis in freshwater test (Emmart & Bates, 1968). Donaldson et al. (1968) using the method of Cole & Li, (1959) produced a preparation of salmon prolactin which was biologically active in maintaining plasma osmolarity of hypophysectomized goldfish kept in fresh water. However, the salmon prolactin was not characterized.
This study was undertaken for the purpose of providing a supply of purified mullet prolactin for radioimmunoassay and to contribute to the sparse knowledge of fish hormones in general. MATERIALS AND METHODS
Preparation of pituitary glands Four hundred adult grey mullet, Mugil cephalus, were netted near the mouth of the Anclote River, at Tarpon Springs, Florida. The pituitary glands were removed from the freshly killed fish and placed in ethanol on dry ice for transport to the laboratory. The glands were cleaned and freeze dried. Prior to use, the dry glands were homogenized with a Potter-Elvehjem homogenizer, centrifuged at 5000 x g for 30 min and the supernatant was lyophilized. The resulting 293 mg of pituitary powder was stored at -10°C until use.
Ultrafiltration One hundred mg of pituitary powder dissolved in 10 ml of 0.01 M phosphate buffer, pH 7-3 containing 0"3 M NaCI (PBS) was placed in a stirred cell and filtered through an XM 50 Diaflo membrane ultrafilterer (Amicon Corp.). This membrane retains material with mol. wt of 50,000 or greater. The ultrafilterer was washed twice with 5 ml of PBS. The ultrafiltrate was next filtered through a UM 2 Diaflo membrane which retains material with mol. wt of 1000 or greater. The ultrafilterer was washed as above. The material remaining on the membrane, consisting of proteins with mol. wt of between 1000 and 50,000 was recovered and dissolved in 2 ml of PBS. Five mg of bovine serum albumin and 5 mg of horse heart cytochrome c were added as mol. wt markers.
Sephadex chromatography
The pituitary powder, mol. wt marker mixture was applied to an 80 cm x 1.5 cm G-75 Sephadex column and chromatographed with PBS at 2°C at a flow rate of 2 ml/ hr. One ml fractions were collected with an automatic fracThis study was supported by the National Science Foun- tion collector. Optical density was determined for each dation Research Grant GB 8019. fraction at 280 nm. 133
134
J.T. WOOSLEYAND J. R. L!xro>4
0.2
E c 0 m
I
o w 0.1 a
-g.
0
!
40
I00
70 Fraction
Number
Fig. 1. Elution profile of mullet pituitary powder mol. wt marker preparation from G-75 Sephadex.
Xanthophore dispersion assay Prolactin has been found to cause dispersion of pigments in xanthophores in Gillichthys mirabilis (Sage, 1970). This discovery was subsequently developed into a sensitive bioassay for fish prolactin by Sage & Bern (1971). Test animals were purchased from a commercial source in San Francisco, California. They were maintained in the laboratory in sea water of 30~L, salinity and at a temperature of 18 20°C. Fractions from the Sephadex chromatography column v~ere examined for biological activity using the Sage & Bern assay as follows. One tenth ml aliquot of each fraction are injected subcutaneously into the preopercular region behind the eye on one side. The same region on the contralateral side was injected with 0"1 ml of PBS as a control. A positive response was recorded when the test side became visibly more yellow than the control side. Fractions containing high levels of fish prolactin were diluted with PBS and the assay repeated. The lowest concentration of material which gave a positive response in at least 50~ of the animals tested was designated as 1 unit of activity. Results were recorded as units Of activity per ml of Sephadex G-75 eluent.
Immunological studies Ovine prolactin antisera were produced in New Zealand white rabbits by a modification of the method of Vaituk-
aitis et al. (1971). One tenth mg of ovine prolactin (NIH P S 10) and 0-1 ml of PBS were immulsified with 0.1 mt Freund's Complete Adjuvant and injected subcutaneously into 30-50 sites on the rabbits backs. One month after the initial injection a booster injection of 0.01 mg of antigen in 0.1 ml PBS and 0.I ml of Freund's Incomplete Adjuvant was given by the same method. The following week an additional booster was given in the same manner. The animals were bled by cardiac puncture during the sixth week. The immunoglobin fraction was isolated from the serum by salt precipitation (Williams & Chase, 1967). The precipitated immunoglobin was diluted to the original volume of antiserum with PBS. The immunoglobins were tested for specificity by double diffusion tests in agar gel against ovine prolactin, ovine STH, Bovine TSH, Procine FSH and bovine LH and extracts of mullet pituitary powder.
Radioimmunoassay Ovine prolactin was radioiodinated with 13~I by the tactoperoxidase method of Marchalonis (1969) as modified by Miyachi et al. (1972). Unbound ~3~I was removed by chromatography on a 0.5cm × 15craG-10 sephadex column which had been washed with 0-05 M phosphate buffer, pH7-3 containing 0.15MNaC1 and I!!~ bovine
0.2
400
E
~ E
a
o m
c
II I
>,
a
0.1 w
/
g. 0
0
20o ._~
7O
4O Fraction
100
Number
Fig. 2. Biological activity of pituitary powder (. . . . ), plotted over the elution profile ( Sephadex G-75.
) fi-om
Prolactin from the grey mullet 10
~
135
÷--'--"--+
0 0 0 X
~gE a. c:
g
u
0 0
i
!
!
1 0 .2
10 °
10 2
•g of Unlabeled Hormone
Fig. 3. Competition between ~3~I labeled Ovine Prolactin (O), mullet pituitary powder (O), and Ovine STH (×). serum albumin. The column was equilibrated and developed with the phosphate buffer. Radioimmunoassays were performed by a modification of the method of Catt & Treagear (1967). Antibody was adsorbed to the inner surface of 10 × 75 mm polystyrene tubes. Four tenths ml of a 1:50 (v/v) of the immunoglobin fraction was added to each tube. The tubes were incubated for 2 hr at 22°C, the contents aspirated, and the tubes washed 3 times with PBS. The tubes were refilled with the same buffer containing 1% bovine serum albumin and incubated an additional 2 hr. The contents were then aspirated and the tubes washed as above. Three tenths ml of 131~ labeled ovine prolactin, in amounts sufficient to saturate the bound antibody, was added to each tube. The tubes were incubated at 22°C for 24 hr and counted to be sure that all tubes contained the same level of radioactivity. The contents were aspirated, the tubes rinsed 3 times with buffer and recounted to establish bound counts per minute. Radioimmunoassays were performed on alternate fractions from the Sephadex G-75 column as follows. One tenth ml of the fractions were added to the tubes and incu-
bated for 24 hr and the per cent binding was determined. Radioimmunoassays were also performed on ovine prolactin, bovine STH, and mullet pituitary powder. RESULTS
Sephadex chromatography Figure 1 shows the elution profile from the G-75 Sephadex column of the mullet pituitary powdermol.wt marker preparation. The largest marker protein (bovine serum albumin) with a mol. wt of 67,000, is seen to elute immediately after the void volume. Following this are several smaller peaks representing components of the pituitary powder. The second large peak is the mol. wt marker cytochrome C with a mol. wt of 12,400.
Bioassay In Fig. 2, the Gillichthys xanthophore dispersion assay results are superimposed upon the sephadex
0.2
~-0"0~0.0~¢ I
E
O O O
I:
0 0
3
0.!
I I
e
g
o.
&
o
Q.
a L
I
o U
%
o
0
X
40
7Q Fraction
100
Number
Fig. 4. Radioimmunoassay results ( - - - -) plotted over the elution profile of mullet pituitary powder from G-75 Sephadex ( ).
130
J.T. WOOSLEVAND J. R. LINTON 2.0
-o
"B >
~4
1.0
5.2
3.6
Log of Molecular Weight
Fig. 5. Determination of tool. wt of mullet "Prolactin." elution profile. Activity is expressed as units per ml of column effluent. It is seen that the peak of biological activity corresponds to a small peak on the elution profile. This peak contains a concentration of 0-11 units per ttg of the original pituitary powder. Radioimmunoassa y
Figure 3 compares the competition between 131~ labeled prolactin, unlabeled ovine prolactin, mullet pituitary powder, and bovine STH, for the antiovine prolacfin antibody. It can be seen that less competition occurred in the case of mullet pituitary powder than with ovine prolactin and that none occurred with bovine STH. The radioimmunoassay data for the Sephadex G-75 fractions are superimposed upon the elution profile in Fig. 4. The highest degree of competition occurred in the fractions which showed biological activity with the Gillichthys assay. In addition, fractions eluting just behind the bovine serum albumin, also compete with 131~ labeled ovine prolactin. Molecular wei,qht estimation
Elution data from the G-75 Sephadex column were analyzed after the method of Whitaker (1963) and shown in Fig. 5. Reference points are provided by the tool. wt markers, bovine serum albumin and cytochrome c. This analysis indicates that mullet prolactin has a minimum mol. wt qf 18,000 and a maximum of 25,000. The peak of biological and immunological activity elutes at a volume corresponding to a mol. wt of 22A00.
DISCUSSION
Prolactin was isolated from the pituitary gland of the grey mullet by initial extraction with phosphate buffered saline. This extraction method preserved both biological and immunological activity of the hormone. Since previous studies in the laboratory indicated that mullet prolactin had a mol. wt similar to that of ovine prolactin it was possible to prepare
extracts for chromatography by selective ultrafiltration. The ultrafiltration and chromatography procedure allowed for both the isolation of the prolactin and the estimation of mol. wt. The estimated mol. wt was 22,400. This is comparable to the average tool. wt of mammalian prolactin (24,000) (Andrews, 1966; Cole et al., 1957; Li et al., 1957}. Measurement of the concentration of mullet prolactin by its biological activity with the Sage & Bern (1971) assay led to an estimate of 0.011 units per ~tg of the original pituitary powder. This is possibly an underestimate since concentrations reported for certain other teleosts range from 100 to 1000 units per #g (Sage & Bern, 1971). Radioimmunoassay of isolated mullet prolactin and pituitary powder showed that the amount of cross reactivity between anti-ovine prolactin antibodies is rather small. One hundred/tg of mullet prolactin was required to displace the same amount of labeled ovine prolactin as was displaced by 5 ng of unlabeled ovine prolactin. Assuming the mullet pituitary powder contained only 1% prolactin, then l#g of mullet prolactin would he immunologically equivalent to only 5 ng of ovine prolactin. However, this degree of sensitivity may be adequate to permit measurement of circulating fish prolactin. Careful examination of the sephadex fractions (Fig. 4) showed that some material with immunological activity eluted from the column just behind the bovine serum albumin marker. This peak may consist of aggregates, possibly dimers of the fish prolactin. It is known that the mammalian hormones have a tendency to form aggregates (Andrews, 1966: Cole & Li, 1958, Ferguson & Wallace. 1960; Reisfeld et al,, 1964; Squire et al., 1960). This aggregation may also account for the apparent underestimation of the concentration of mullet prolactin in the pituitary powder. The similarity in mol. wt and the occurrence of immunological reactivity between mullet prolactin and ovine prolactin provides some evidence to support the speculation that mammalian prolactins have evolved from the prolactin-like hormones of lower vertebrates (Nico!! & Bern, 1968).
Prolactin from the grey mullet REFERENCES
ANDgEWS P. (1966) Molecular weights of prolactins and pituitary growth hormones estimated by gel filtration. Nature, Lond. 209, 155--157. BARESR. W. & RIDDLE O. (1935) The preparation of prolactin. J. Pharmac. exp. Ther. 55, 365-371. CArt K. & TREGEARG. W. (1967) Solid-phase radioimmunoassay in antibody-coated tubes. Science, N.Y. 158, 1570-1573. Cr~AOWICK A. (1965) Prolactin-like activity in extracts of fish pituitaries. Gen. & compar. Endocr. 5, 668-669. (Abstr.). COLE R. D., GESCHWlNDI. I. & LI C. H. (1957) Studies on pituitary lactogenic hormone--XV. N-Terminal residue analysis and n-terminal sequence analysis. J. biol. Chem. 224, 399-405. COLE R. D. & LI C. H. (1959) Occurrence of several active components in ovine lactogenic hormone as revealed by zone electrophoresis on starch. Biochim. biophys. Acta 33, 563-564. DONALDSONE. M., YAMAZAKIF. & CLARKEW. C. (1968) Effects of hypophysectomy on plasma osmolarity in goldfish and its reversal by ovine prolactin and a preparation of salomon pituitary "prolactin". J. Fish. Res. Bd Can. 25, 1497-1500. EMMARTE. W. & BATESR. W. (1968) The use of immunochemical binding and Sephadex filtration in procedures toward the purification of ovine and piseine "prolactin'. Gen. & compar. Endocr. 11, 580-594. EMMARTE. W., PICKFORDG. E. & WILHELMIA. E. (1966) Localization of prolactin within the pituitary of a cyprinodont fish, Fundulus heteroclitus (Linnaeus) by a specific fluorescent antiovine prolactin globulin. Gen. & compar. Endocr. 7, 571-583. EMMARTE. W. & WILHELM1A. E. (1968) Immunochemical studies with prolactin-like fractions of fish pituitaries. Gen. & compar. Endocr. 11, 515-527. FERGUSON K. A. & WALLACEA. S. C. (1960) Starch gel electrophoresis of anterior pituitary hormones. Acta Endocr. Suppl. 51, 293. (Abstr.). LI C. H., COLE R. D. & CORALM. (1957) Studies on pituitary lactogenic hormone--XVI. Molecular weight of the ovine hormone. J. biol. Chem. 229, 153-156.
137
L~ C. H., S1MPSO~ M. E. & EVANS H. M. (1942) Studies on pituitary lactogenic hormone--VII. A method of isolation. J. biol. Chem. 146, 627-631. MARCHALONISJ. J. (1969) An enzymic method for the trace iodination of immunoglobulins and other proteins. Biochem. J. I13, 299-305. MIYACHI Y., VAITUKAITIS,J. L., NIESCHLAGE. & LIPSETr M. B. (1972) Enzymatic radioiodinations of gonadotropins. J. clin. Endocr. Metab. 34, 23-28. NICOLL C. S. & BERN H. A. (1968) Further analysis of the occurrence of pigeon crop sac-stimulating activity (prolactin) in the vertebrate adenohypophysis. Gen. & compar. Endocr. 11, 5-20. PICKFORDG. E., ROBERTSONE. E. & SAWYERW. H. (1965) Hypophysectomy, replacement therapy and the tolerance of the euryhaline killifish, Fundulus heteroclitus, to hypotonic media. Gen. & compar. Endocr. 5, 160-180. REISFELD R. A., WILLIAMS D. E. C1RILLO V. J. TONG G. L. & BRI~K N. G. (1964) Characterization of sheep prolactin. J. biol. Chem. 239, 1777-1782. SAGE M. (1970) Control of prolactin release and its role in color change in the teleost Gillichthys mirabilis. J. exp. Zool. 173, 121-128. SAGEM. & BERN H. A. (1971) Assay of prolactin in vertebrate pituitaries by its despersion of xanthophore pigment in the teleost GiUichthys mirabilis. J. exp. Zool. 180, 169-174. SQUIRE P. S., STARMANB. & LK C. H. (1963) Studies on pituitary lactogenic hormone--XXII. Analysis of the state of aggregation of the ovine hormone by ultracentrifugation and exclusion chromatography. J. biol. Chem. 238, 1389-1395. VAITUKAITISJ., ROBBINS J. B., NIESCHLAGE. & ROSS G. T. (1971) A method for producing specific antisera with small doses of immunogen. J. clin. Endocr. Metab. 33, 988-991. WH1TAKERJ. R. (1963) Determination of molecular weights of proteins by gel filtration of Sephadex. Amdyt. Chem. 35, 1950-1953. WILLIAMSC. A. & CHASE M. W. (19671 Methods in Immunology and Immunochemistry. Vol. I. Academic Press, New York.
ISOLATION AND CHARACTERIZATION OF PROLACTIN FROM THE GREY MULLET, M U G I L CEPHALUS J. T. WOOSLEY AND J. R. LrNTON Department of Biology, University of South Florida, Tampa, FL 33620, U.S.A.
(Received 21 May 1974) Abstract--1. A prolactin-like hormone has been isolated from the pituitary of the grey mullet, Mugil cephalus (Linnaeus), by ultrafiltration and analytical chromatography on G-75 Sephadex. 2. The hormone possessed biological activity in the Gillichthys xanthophore dispersion assay (Sage & Bern, 1971). 3. A sensitive radioimmunoassay for mullet prolactin was developed and validated by comparison with biological activity data. Some immunological similarity between the ovine prolactin molecule and the mullet prolactin molecule was demonstrated by this radioimmunoassay. 4. A molecular weight of 22,400 was indicated by the elution behavior of mullet prolactin from G-75 Sephadex.
INTRODUCTION
THERE have been several attempts to isolate fish prolactin. Chadwick (1965), applied Sephadex chromatography and starch gel electrophoresis to pituitary extracts of several teleosts and elasmobranch species. Activity was measured by the mammary gland and pigeon crop sac bioassays. Little activity was found and no attempt was made to characterize the fish hormone. Pickford et al. (1965) extracted carp pituitaries using a modification of the Bates & Riddle (1935) procedure. The isolated material did not have biological activity as measured by its ability to promote survival of hypophysectomized Fundulus heteroclitis in fresh water. Material from carp and pollack pituitaries has been found to cross react with ovine and bovine prolactin antibodies (Emmart et al., 1966, Emmart & Wilhelmi, 1968). This finding was exploited in an attempt to isolate fish prolactin by Emmart & Bates (1968). Anti-ovine prolactin antibodies were added to fractions from carp and pollack pituitaries. The precipitated antigen-antibody complex was collected, dissolved, and chromatographed on G-200 sephadex. Several broad peaks were obtained which reacted immunologically with antiovine antibodies. These results indicate that fish prolactin may form aggregates at low pH as does the mammalian hormone (Andrews 1966; Cole & Li, 1959; Ferguson & Wallace, 1960; Riesfeld et al., 1964; Squire et al., 1963). The material isolated in this manner did not have biological activity using the survival of F. heteroclitis in freshwater test (Emmart & Bates, 1968). Donaldson et al. (1968) using the method of Cole & Li, (1959) produced a preparation of salmon prolactin which was biologically active in maintaining plasma osmolarity of hypophysectomized goldfish kept in fresh water. However, the salmon prolactin was not characterized.
This study was undertaken for the purpose of providing a supply of purified mullet prolactin for radioimmunoassay and to contribute to the sparse knowledge of fish hormones in general. MATERIALS AND METHODS
Preparation of pituitary glands Four hundred adult grey mullet, Mugil cephalus, were netted near the mouth of the Anclote River, at Tarpon Springs, Florida. The pituitary glands were removed from the freshly killed fish and placed in ethanol on dry ice for transport to the laboratory. The glands were cleaned and freeze dried. Prior to use, the dry glands were homogenized with a Potter-Elvehjem homogenizer, centrifuged at 5000 x g for 30 min and the supernatant was lyophilized. The resulting 293 mg of pituitary powder was stored at -10°C until use.
Ultrafiltration One hundred mg of pituitary powder dissolved in 10 ml of 0.01 M phosphate buffer, pH 7-3 containing 0"3 M NaCI (PBS) was placed in a stirred cell and filtered through an XM 50 Diaflo membrane ultrafilterer (Amicon Corp.). This membrane retains material with mol. wt of 50,000 or greater. The ultrafilterer was washed twice with 5 ml of PBS. The ultrafiltrate was next filtered through a UM 2 Diaflo membrane which retains material with mol. wt of 1000 or greater. The ultrafilterer was washed as above. The material remaining on the membrane, consisting of proteins with mol. wt of between 1000 and 50,000 was recovered and dissolved in 2 ml of PBS. Five mg of bovine serum albumin and 5 mg of horse heart cytochrome c were added as mol. wt markers.
Sephadex chromatography
The pituitary powder, mol. wt marker mixture was applied to an 80 cm x 1.5 cm G-75 Sephadex column and chromatographed with PBS at 2°C at a flow rate of 2 ml/ hr. One ml fractions were collected with an automatic fracThis study was supported by the National Science Foun- tion collector. Optical density was determined for each dation Research Grant GB 8019. fraction at 280 nm. 133
134
J.T. WOOSLEYAND J. R. L!xro>4
0.2
E c 0 m
I
o w 0.1 a
-g.
0
!
40
I00
70 Fraction
Number
Fig. 1. Elution profile of mullet pituitary powder mol. wt marker preparation from G-75 Sephadex.
Xanthophore dispersion assay Prolactin has been found to cause dispersion of pigments in xanthophores in Gillichthys mirabilis (Sage, 1970). This discovery was subsequently developed into a sensitive bioassay for fish prolactin by Sage & Bern (1971). Test animals were purchased from a commercial source in San Francisco, California. They were maintained in the laboratory in sea water of 30~L, salinity and at a temperature of 18 20°C. Fractions from the Sephadex chromatography column v~ere examined for biological activity using the Sage & Bern assay as follows. One tenth ml aliquot of each fraction are injected subcutaneously into the preopercular region behind the eye on one side. The same region on the contralateral side was injected with 0"1 ml of PBS as a control. A positive response was recorded when the test side became visibly more yellow than the control side. Fractions containing high levels of fish prolactin were diluted with PBS and the assay repeated. The lowest concentration of material which gave a positive response in at least 50~ of the animals tested was designated as 1 unit of activity. Results were recorded as units Of activity per ml of Sephadex G-75 eluent.
Immunological studies Ovine prolactin antisera were produced in New Zealand white rabbits by a modification of the method of Vaituk-
aitis et al. (1971). One tenth mg of ovine prolactin (NIH P S 10) and 0-1 ml of PBS were immulsified with 0.1 mt Freund's Complete Adjuvant and injected subcutaneously into 30-50 sites on the rabbits backs. One month after the initial injection a booster injection of 0.01 mg of antigen in 0.1 ml PBS and 0.I ml of Freund's Incomplete Adjuvant was given by the same method. The following week an additional booster was given in the same manner. The animals were bled by cardiac puncture during the sixth week. The immunoglobin fraction was isolated from the serum by salt precipitation (Williams & Chase, 1967). The precipitated immunoglobin was diluted to the original volume of antiserum with PBS. The immunoglobins were tested for specificity by double diffusion tests in agar gel against ovine prolactin, ovine STH, Bovine TSH, Procine FSH and bovine LH and extracts of mullet pituitary powder.
Radioimmunoassay Ovine prolactin was radioiodinated with 13~I by the tactoperoxidase method of Marchalonis (1969) as modified by Miyachi et al. (1972). Unbound ~3~I was removed by chromatography on a 0.5cm × 15craG-10 sephadex column which had been washed with 0-05 M phosphate buffer, pH7-3 containing 0.15MNaC1 and I!!~ bovine
0.2
400
E
~ E
a
o m
c
II I
>,
a
0.1 w
/
g. 0
0
20o ._~
7O
4O Fraction
100
Number
Fig. 2. Biological activity of pituitary powder (. . . . ), plotted over the elution profile ( Sephadex G-75.
) fi-om
Prolactin from the grey mullet 10
~
135
÷--'--"--+
0 0 0 X
~gE a. c:
g
u
0 0
i
!
!
1 0 .2
10 °
10 2
•g of Unlabeled Hormone
Fig. 3. Competition between ~3~I labeled Ovine Prolactin (O), mullet pituitary powder (O), and Ovine STH (×). serum albumin. The column was equilibrated and developed with the phosphate buffer. Radioimmunoassays were performed by a modification of the method of Catt & Treagear (1967). Antibody was adsorbed to the inner surface of 10 × 75 mm polystyrene tubes. Four tenths ml of a 1:50 (v/v) of the immunoglobin fraction was added to each tube. The tubes were incubated for 2 hr at 22°C, the contents aspirated, and the tubes washed 3 times with PBS. The tubes were refilled with the same buffer containing 1% bovine serum albumin and incubated an additional 2 hr. The contents were then aspirated and the tubes washed as above. Three tenths ml of 131~ labeled ovine prolactin, in amounts sufficient to saturate the bound antibody, was added to each tube. The tubes were incubated at 22°C for 24 hr and counted to be sure that all tubes contained the same level of radioactivity. The contents were aspirated, the tubes rinsed 3 times with buffer and recounted to establish bound counts per minute. Radioimmunoassays were performed on alternate fractions from the Sephadex G-75 column as follows. One tenth ml of the fractions were added to the tubes and incu-
bated for 24 hr and the per cent binding was determined. Radioimmunoassays were also performed on ovine prolactin, bovine STH, and mullet pituitary powder. RESULTS
Sephadex chromatography Figure 1 shows the elution profile from the G-75 Sephadex column of the mullet pituitary powdermol.wt marker preparation. The largest marker protein (bovine serum albumin) with a mol. wt of 67,000, is seen to elute immediately after the void volume. Following this are several smaller peaks representing components of the pituitary powder. The second large peak is the mol. wt marker cytochrome C with a mol. wt of 12,400.
Bioassay In Fig. 2, the Gillichthys xanthophore dispersion assay results are superimposed upon the sephadex
0.2
~-0"0~0.0~¢ I
E
O O O
I:
0 0
3
0.!
I I
e
g
o.
&
o
Q.
a L
I
o U
%
o
0
X
40
7Q Fraction
100
Number
Fig. 4. Radioimmunoassay results ( - - - -) plotted over the elution profile of mullet pituitary powder from G-75 Sephadex ( ).
130
J.T. WOOSLEVAND J. R. LINTON 2.0
-o
"B >
~4
1.0
5.2
3.6
Log of Molecular Weight
Fig. 5. Determination of tool. wt of mullet "Prolactin." elution profile. Activity is expressed as units per ml of column effluent. It is seen that the peak of biological activity corresponds to a small peak on the elution profile. This peak contains a concentration of 0-11 units per ttg of the original pituitary powder. Radioimmunoassa y
Figure 3 compares the competition between 131~ labeled prolactin, unlabeled ovine prolactin, mullet pituitary powder, and bovine STH, for the antiovine prolacfin antibody. It can be seen that less competition occurred in the case of mullet pituitary powder than with ovine prolactin and that none occurred with bovine STH. The radioimmunoassay data for the Sephadex G-75 fractions are superimposed upon the elution profile in Fig. 4. The highest degree of competition occurred in the fractions which showed biological activity with the Gillichthys assay. In addition, fractions eluting just behind the bovine serum albumin, also compete with 131~ labeled ovine prolactin. Molecular wei,qht estimation
Elution data from the G-75 Sephadex column were analyzed after the method of Whitaker (1963) and shown in Fig. 5. Reference points are provided by the tool. wt markers, bovine serum albumin and cytochrome c. This analysis indicates that mullet prolactin has a minimum mol. wt qf 18,000 and a maximum of 25,000. The peak of biological and immunological activity elutes at a volume corresponding to a mol. wt of 22A00.
DISCUSSION
Prolactin was isolated from the pituitary gland of the grey mullet by initial extraction with phosphate buffered saline. This extraction method preserved both biological and immunological activity of the hormone. Since previous studies in the laboratory indicated that mullet prolactin had a mol. wt similar to that of ovine prolactin it was possible to prepare
extracts for chromatography by selective ultrafiltration. The ultrafiltration and chromatography procedure allowed for both the isolation of the prolactin and the estimation of mol. wt. The estimated mol. wt was 22,400. This is comparable to the average tool. wt of mammalian prolactin (24,000) (Andrews, 1966; Cole et al., 1957; Li et al., 1957}. Measurement of the concentration of mullet prolactin by its biological activity with the Sage & Bern (1971) assay led to an estimate of 0.011 units per ~tg of the original pituitary powder. This is possibly an underestimate since concentrations reported for certain other teleosts range from 100 to 1000 units per #g (Sage & Bern, 1971). Radioimmunoassay of isolated mullet prolactin and pituitary powder showed that the amount of cross reactivity between anti-ovine prolactin antibodies is rather small. One hundred/tg of mullet prolactin was required to displace the same amount of labeled ovine prolactin as was displaced by 5 ng of unlabeled ovine prolactin. Assuming the mullet pituitary powder contained only 1% prolactin, then l#g of mullet prolactin would he immunologically equivalent to only 5 ng of ovine prolactin. However, this degree of sensitivity may be adequate to permit measurement of circulating fish prolactin. Careful examination of the sephadex fractions (Fig. 4) showed that some material with immunological activity eluted from the column just behind the bovine serum albumin marker. This peak may consist of aggregates, possibly dimers of the fish prolactin. It is known that the mammalian hormones have a tendency to form aggregates (Andrews, 1966: Cole & Li, 1958, Ferguson & Wallace. 1960; Reisfeld et al,, 1964; Squire et al., 1960). This aggregation may also account for the apparent underestimation of the concentration of mullet prolactin in the pituitary powder. The similarity in mol. wt and the occurrence of immunological reactivity between mullet prolactin and ovine prolactin provides some evidence to support the speculation that mammalian prolactins have evolved from the prolactin-like hormones of lower vertebrates (Nico!! & Bern, 1968).
Prolactin from the grey mullet REFERENCES
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