Life Scieac~s Vol . 18, pp . 245-250 Printed in Che û.S .A .
ISOLATION AND CHARACTERIZATION OF LUTEINIZING HORMONE FROM AMPHIBIAN ( RANA CATESBEIANA) PITUITARIES Harold Papkoffl~ 2, Susan Walker Farmerl, and Paul Licht 3
1 Hormone Research Laboratory and 2Reproductive Endocrinology Center ~Jniversity of California, Saa Francisco, Calif.94143 and s Dept . of Zoology, University of California, Berkeley, California, 94720 (Received in final form January 2, 1976) SUMMARY: We report here the first isolation of . an anterior pituitary~ormone from an amphibian species, the bullfrog ( Rang catesbeiana) . HIghly purified luteinizing hormone was isolated from alkaline entracte of bullfrog pituitaries by salt fractionation, chromatography on ion-eachangers and gel filtration . Characterisation etudiee show the hormone to contain 9% carbohydrate and to possess sa amino acid composition similar to ovine luteinizing hormone. Sedimentation-velocity experiments in the ultracentrifuge indicate that the bullfrog gonadotropin dissociates in acidic solution and ie composed of eubuaite . Bullfrog luteinizing hormone ie highly active is as in vitro toad ovulation assay and also ellicite testosterone production iâvitro from isolated rat testis Leydig cells . INTRODUCTION The preparation and properties of a number of mammalian species of luteinizing hormone (interstitial cell stimulating hormone, LH, ICSH) have been extensively described (see (1) for a recent review). In contrast, there ie little definitive biochemical data on non-mammalian forms of this goaadotropia, and in particular, none whatsoever on Amphibian pituitary gonadotropine . HIaderiag the study of non-mammalian gonadotropins has been the insensitivity of standard mammalian gonadotropia bioassays to these materials as well ae the problem of acquiring sufficient quantities of pituitaries to affect isolation etudiee . We have recently shown (2), employing non-mammalian tact systems, that the bullfrog ( Raga cateebeiana ) pituitary possesses two distinct gonadotropins resembling mammaliân LI~and FSH. The bullfrog ie of special interest since it represents a vertebrate species more primitive than birds and reptiles . In this communication, we report the isolation of bullfrog LH is highly purified form . As far se we are aware, this ie the first report on the characterisation of an anterior pituitary hormone isolated from an amphibian species. EXPERIMENTAL RESULTS Purification . Approximately two tone of fresh frozen bullfrog heads (about 1 0,~ OÔO) were obtained from commercial sources in Japan. The pituitaries were dissected over a period of a year and stored at -20 ° until used . Approximately 890 gm of pituitary tissue was obtained . The material 245
Bullfrog Luteiaiziag Hormone
Vol . 18, No . 2
was processed in batches of 200-400 gm . The initial steps of the purification have been described in detail in our previous report (Z). In brie£, the tissue was homogenized with cold water in a Waring Blendor, adjusted to pH 9 . 5 with Ca0 and stirred for Z-3 hre . Following centrifugation, the extract was adjusted to 0.6 saturated (NH4)ZS04(3g0. g/1) . The resultant precipate (Z9 . 3 gm) was chromatographed on columns of Amberlite CG-50 as previously described and yielded 8 . 8 gm in the LH fraction (Fraction C, pH 6 . 0) . Further purification was achieved by a minor modification (use of NH4HC03 buffers) of the sulfoethyl-Sephadex, C50 chromatographic system previously employed for ovine sad bovine LH (3,4). At thin stage the absorbed LH fraction amounted to l . 76 g . Final purification was achieved by passing the above material over DEAE-cellulose in 0. 03M NH4HC03 , pH 9. 0 as described before (Z), and by gel-filtration on columns of Sepbadex G-100 in 0 . 05M NH~HC0 3. The major peak, emerging with a Ve/Vo of 1, 8-1 .9 contained the LH and was refiltered on Sephadex G-100 to obtain the final purified preparation. From 890 gm of pituitary tissue, approximately 350 mg of purified LH wan obtained . Biological Activities . The purified bullfrog LH was assayed by the in vitro ovulation assay employing segments of ovaries obtained from the tome Xenopus laevie (5). Repeated assays show the material to average 0 . 3 x NIH LH- 1 range, 0 . Z-0. 7) . The potency of the pituitary extract used for purification was 0. 06 x NIH-LH-S1 . FSH contamination was assessed by testing is the hypophyaectomized male lizard, Anolis carolineneie, as previously described (6). The bullfrog LH was found tome an activity equivalent to 0 . 1 x NIH-FSH-S1, which was judged to be low insofar as partially purified bullfrog FSH has been obtained with an activity of 5-10 x NIH-FSH-S10 (see also ref. Z) . This low potency in Anolie assay may, in fact, represent intrinsic activity of the LH molecule~7 .-preliminary tests for TSH activity (using thyroidal radioiodine uptake in young bullfrogs ) indicate a relatively low TSH contamination in the bullfrog LH. Bullfrog LH has also been shown to stimulate the in vitro production of testosterone by minced rabbit testis (8) and in the present s~-y, similar results were obtained with isolated rat Leydig cells employing the conditions described by Ramachandran and Sairam (9). Figure 1 shows the response ellicited by bullfrog LH compared to ovine LH. It can be seen that the bullfrog LH is similar to ovine LH in terms of the dose-response elope and in the maximal production of testosterone ; however, the bullfrog LH is considerably lees potent, requiring about 1000 x the amount of ovine LH to effect similar results . Similar results were obtained with the rabbit testis tisane (8). Physical sad Chemical Characterization , Purified bullfrog LH behaves on gel filtration columns much like mammalian LH and emerges from Sephadex G-100 columns with a similar Ve/Vo (1 .8-1 . 9) . The material has been examined by disc gel electrophoresis on columns of polyacrylamide at pH 8, 3 and pH 4. 5. At the former pH no stainable bands were detected suggesting the material does not enter the gel at this pH . At pH 4. 5, however, two clearly stained bands were observed with relatively low mobility . Similar patterns have been previously observed with ovine LH (10) .
Bullfrog Luteiniziag Hormone
Vol. 18, No . 2
Bullfrog LH was examined in the Spinco Model E ultracentrifuge for it~ sedimenting properties at 59, 780 r. p. m. Mondodisperse boundaries were observed at pH 7. 1 (0 . O1M phosphate - 0, lOM NaCl) and a sedimentation coefficient of 2.48 S was calculated . At pH 1 . 5 (0 . 2M NaCl-HCl), a value of 1 . 80 S was obtained which ie as indication of dissociation like the original observations made showing the subunit nature of ovine LH (11) . Carbohydrate analysis by the colorimetric methods previously employed (3,4), revealed that bullfrog LH possesses 3.6°fs hexose, 5.5°J. heaoeamine, and a small amount (0 . 2°f. ) of sialic acid. The total of 9. 3°f. carbohydrate is about 60°J. of that obtained for ovine LH (Table 1) . TABLE 1 Carbohydrate Content* of Bullfrog LH Compared to Sheep LH Sugar
Hexoee Hexoeamine Sialic Acid
Bullfrog 3.6 5.5 0. 2
Sheep 7 .Z 9.1 0.4
* Eapreseed se g/100 g glycoprotein ; uncorrected for moisture and ash. TABLE 2 Amino Acid Content of Bullfrog LH Compared to Sheep LH Amino Acid
Lysine Histidine Arginine Aspartic Threonine Serine Glutamic Proline Glycine Alanine Half-cyetine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophane
Bullfrogs 13 . 3 5.7 9. 0 Z3 . 9 21 .3 16 .8 14 . 5 15 .8 7.5 9. 6 21 .5 1 Z. 0 6.9 11 .6 11 . 6 8.3 6.4 0 .9 c
Sheepb 12 6 11 11 16 14 14 27 11 15 tt 13 7 7 14 7 8 0
a20 hr . hydrolysis, results calculated as residues/215 residues analyzed . bCalculated from the amino acid sequences of sheep LH subunits (17 ) . °Determined spectrophometrically and calculated se residues/30000 g .
Bullfrog Luteiniziag Hormone
Vol. 18, No . 2
The amino acid content of bullfrog LH determined by the method of Spackman et al . (1Z) is seen in Table Z. When the values were compared with that for tieôvine hormone, many similarities are seen, especially with respect to the content of the basic amino acids, the half-cystine content, the aromatic amino acids, as well as several others . Notable differences are seen in the aspartic acid content (Z4 vs 11 for ovine) and in the lower content of proliae (16 vs Z7 for ovine) . In addition, spectrophotometric analysis suggests the presence of tryptophan in bullfrog LH whereas it is absent in ovine LH. Amino terminal group analysis was performed by the Dansyl technique (13) and showed phenylalanine to be the major terminal amino acid as well as the presence of lesser amounts of leucine and threonine. DLS CUSSION This work has resulted in the preparation of highly purified LH in good yield from bullfrog pituitaries such that meaningful chemical and biological studies can be undertaken to explore problems of gonadotropin action, species-specificity, and the evolution of hormone structures . It is of interest that by and large the methodology of preparation are those which have been found to be successful with n~+~+++*r,nt iaa pituitary tissue and more recently with avian (14) and reptillisn pituitaries (5, 15). Studies is progress with diverse vertebrate species suggest that the methods we have employed may have a general applicability. Preliminary characterization studies show that the bullfrog LH ie of a similar molecular size to mammalian LH se evidenced by gel-filtration behavior and by its sedimentation properties in the ultracentrifuge. In addition, the latter studies clearly show that bullfrog dissociates in acidic solution (i . e. lowering of SZO~ a, value) and probably consists of subuaite as is the case for ovine LH and other mammalian glycoprotein hormones . We have reported similar evidence for turkey and snapping turtle LH (14, 16) . Other studies reported here show that the bullfrog LH is a glycoprotein (9 . 3°~. carbohydrate) and has an amino acid content which is similar in many respects to ovine LH. The differences found, however, point to structural variations and will undoubtedly be related to the species-specificity exhibited by the hormone. With respect to biologicâl activity, the potency obtained in the in vitro ovulation test, 0 . 3 x NIH-LH-S1 is within the range of potencies we hâve obtained with a wide variety of comparably purified mammalian and non mammalian LH's (5, 8, 14, 16). Finally, the activity of the purified bullfrog LH in stimulating the in vitro production o~testoeterone by isolated rat Leydig cells, (Fig . 1)while low, is of interest in terms of suggesting that despite evolution in both hormone and receptor structures, sufficient conservation of structure has been maintained to allow the hormone specific response to be observed . ACKNaWLEDGEMENTS We thank Professor Choh Hao Li for reading the manuscript and his valuable suggestions. The technical assistance of Carol Hopkins, Jean Knorr, Dan Gordon and Alaa Suyama is appreciated . This work was supported in part by grants from the National Science Foundation, GB35Z41X, and The Rockefeller Foundation .
Vol. 18, No . 2
Bullfrog Luteinising Hormone
Stimulation of testosterone production in isolated rat testis Leydig celle by sheep and bullfrog LH . Note different concentration range employed for each. REFERENCES
2. 3. 4. 5. 6. 7. 8. 9.
M. R. Sairam and H. Papkoff in Handbook of Physiology, E. Knobil and W. H . Sawyer, ede . Am. P yeiol . Soc. , Was ngton, D. C. , Sec. 7, Vol. III, Part 2, p~p,lll-131 (1974) . P. Licht and H. Papkoff, Endocrinology 94, 1587-1594 (1974) . H: Papkoff, D. Gespodarowicz, A. Candiotti and C . H. Li, Arch . Biochem. Biophye . _111, 431-438 (1965) . H. Papkoff and J. Gan, Arch . Biochem. Biophye . 136, 522-528 (1970) . P. Licht and H. Papkoff, en, mp . Endocrinol , ï18-237 (19?4) . P. Licht and H. Papkoff, Gen. Comp. Endocrinol . 19, 102-114 (1971) . P. Licht and H. Papkoff, Gen. Comp . Endocrinol . 20, 17Z-176 (1973) . P. Licht, C. Muller and H. W. Teui, Biol . Reprod . ~n prose, 1976) . J. Ramachandran and M. R. Sairam, Arch . Biochem. Biophye . _167, 294-300 (1975) . L. E. Reichert and A. F. Parlow, Endocrinology 73, 285-193(1963). C. H. Li and B. Starman, Nature 20 , 291-292 (1964) .
250 12 . 13 . 14 . 15 . 16 . 17 .
Bullfrog Luteinizing Hormoae
Vol . 18, No . 2
D. H. Spackman, W. H. Stein and S . Moore, Anal . Chem. 30, 11901206 (1958) . W . R. Gray and B. S. Hartley, Biochem. J. _89, 59p; K . R. Wood and K . T . Wang, Biochim. Biophys . Acta 133, 369-370 (1967) . S. W . Farmer, H. Pap off and P. Licit, - Biol . Reprod ._12, 415-4Z2 (1975) : P. Licht, S. W. Farmer and H. Papkoff, Biol . Reprod . (in press,1976) P. Licht and H. Papkoff in Gonadotropins and Gonadal Function , N. R . Moudgal, ed . Acad . Press, New York, pp 101-117 (1974) . H. Papkoff, M. R. Sairam, S. W. Farmer and C. H. Li, Recent Progress in Hormone Research Z9, 563-588 (19?3) .