Life Scisacae, Vol . 25, pp . 541-546 Printed in the II .S .A .
Pergaawn Preas
PRESENCE OF IMMUNOREACTIVE a-ENDORPHIN IN RAT P1TUIi'ARY C3LAND Jtitnichi Fukata, Yoehikatsu Nalrai and Hiroo Imura god Division, Department of hiternal Medicine, Kyoto University Faculty of Medicine, Kyoto 808, Japan. (Received in final fors July 2, 1979)
Utilizing radioimmunoassay for a-e~ndoiphin, we attempted to demnonetrate immunoreactive a-endorphin in acid eatracte of pare distalis a~ oomhined pans intermedia and parr nervosa aS the rat P 9.Blaad attar chromatography on Sephadex Gr2ä ßLipotropin, ~-endorphin a~ y-endorphin orere not converted i~o aemdorphia during the extraction and gel chromatographic procedures . Ca~noemtrations of immunoreactlve a-endorphin determined after gel chromatography of e~dr8ots from pare distalir and combined pare intsrmedis and parr nervosa were L 1±0, 8 and 130±1? ng/mg ~ throe (mean±SE), respectively" ~ Serial dilution of these e~dracts gave parallel lines to the standard curve a~f synthetic a-endbrphin, but not to that of y-endorphin or 6-endorphin. These results suggest the existence of immunoreactive a-endorphin indistinguishable in molecular size from syntheüc a-endorphin in the rat pituitary gland. Boon after the etiuoture of enkephalins wan identified from porcine brain (1), three opioid peptides, a-endorphin, ß-endorphin and Y-esdoiphtn were isolated from pituitary tirsue or the bypothalanno-pituitary comple~c (2, 3) " Recently, the oonoentra bona of ~-endorphin (4, 6) and Y-endorphin (8) in rat pituitary glatds have been measured by radioimmumorrray and the existence of a-endorphin, another opioid peptide, in the rat pituitary glad has bees suggested by Hlocmn et al who used the ianmtmooytofluoreroenoe technique (?). To our lmowledge, however, tbere has been no report caacerntag the concentration of a-endorphin in rat pituitary gland. The present rtud9 was designed ~ demonstrate the exirtemoe of a-endorphin and to measure its concentration in rat Pituitary glaada utilizitg radioimm~moassgy for aendorphin. Materials sad Meth Extraction of a-Endorphin. Three male Wistar rats weighing 400-860 g were decapitated. Pare distalis and combined pare intermedia and pans nervosa of the pituitary gland were carefully separated, weighed and immersed in 2 ml of chilled 0. 2N HCi. All the procedures were completed within one or two minutes. The tisane was boiled for 10 min at 96'C, then immediately cooled and homogenized üpr 90 sec with a Polyfron homogenizer. After centrifhgation at 10, OOOsg in a reirlgerated centxlfhge 0024-3205/79/060541-06$02 .00/0 Copyright (c) 1979 Perganon Press Ltd
54 2
a-Endorphin in Rat Pituitary Gland
Vol . 25, No . 6, 1979
for 30 min, the supernatant was collected ànd lyophilized. The lyophiilized materials were the reconstituted in 0. 06M phosphate buffer (pH 7 . 4) containing 0. 5 ~ human serum albumin, 500 KIU/ml Trasylol and 0. 4 ~ meroaptoethaaol (standard diluent). Recovery of immunorea+ctive a-endorphin during this extraction procedure, as monitored by the addition of 80 ng syntheric a-endorphin to 100 mg of rat liver rissue, was 53 ~. Gel Exclusion Chromatography. A 0. 2 ml aliquot of the reconsrituted solution or a syntheric a-emlorphin was applied on a 0. 7 x 57 om column of 8ephadex G-25 equilibrated and eluted with the standard diluent at 4'C . The flow rate was 1. 5 mI/h and the fraction volume was 0. 74 ml . The a-endorphin content of each fraction was 125I_ measured by radioimmunoassay . Blue dextrsa was used as a marker for Vo, ßh-endorphin (Li) for ß-endorphin sad 12bI for salt peak. Syntheric a-endorphin was used as a marker for a-endorphin, because 125I_a-endorphin eluted at a position slightly behind synthetic a-endorphin . Radioimmunoassay . Radioimmunoassay of a-endorphin was performed using antibody to synthetic a-endorphin (kindly provided by Dr . R. (~uillemin) at the final concentration of 1 :21, 000 . Synthetic a-endorphin was used ae a reference standard and for labeling . a-Endorphia was iodinated with Na 125I using the Chloramine T method of Hunter aml Greenwood (8) . Purlücation of 125I_a-eadorphin was carried out by chromatography oa 3ephadex G-25. The specinc acrivriy of 1251-a-endorphin ranged from 180 to 180 uCi/Rg. Radioimmunoassay was performed by the double antibody immunopreoipitarion technique, utilizing anti-rabbit y-globulin aarisera as the second antibody. The minimal detectable quality of a-endorphin was 20 pg" Ira- and interassay coefficie~s of variation were 3. 4 % and 8. 3 ~, respectively . This antiserum showed weak cross-reactivity with ß-lipotropin, ß-endorphin, y-endorphin and d -endorphin . Hut, Met~enkephalin, heu 5-~kephalin, ß-MSH and human ACTH failed to displace 125 1-a-endorphin from the antiserum eveâ when quantities as large as 10 ng were added (Fig. 1) . Radioimmumassay of ACTH was performed with the antiserum provided by the National Pituitary Agency (9) . Stability of B-endorphin, v-~dorohin and 8-lipotropin during extraction procedure . To exclude the possibility of the conversion of ß-endorphin into a-endorphin in the process of extraction, 2. 4 l+g of sy~heric ß-endorphin was added to combined pans intermedia and pars nervosa of rat pituitaries immediately after separation, boiled and homogenized in 2 ml of chilled 0 . 2N HCI, ~ then extracted and reconsrituted in standard diluent. The preparation was then subjected to radioimmunoasasy for aeadorphin and ACTH. There was no conversion of ß-endorphin into a-endorphin during the extracrion procedure, 88 the ratios of a-endorphin to ACTH concentration from the pituitary extract with or without addition of 2. 4 ug of ß-endorphin were identical (Fig. 2) . In the same way, there was ao apparent conversion of ß-lipotropin . or y-endorphin into a-endorphin . Results The elution profiles of para distalis and combined pars intermedia and pars nervosa of rat pituitary gland are shown in Fig. 3. Ia the extract of pars distalis, two peaks with a-endorphin-like immunoreactivity were found; the first peak eluted in a poeirion compatible with 1251-ß_endorphin and the 2nd peak emerged at the elurioa position of synthetic a-endorphin (designated as immunoreactive a-endorphin hereafter) . Ia extract of combined para intermedia and pars nervosa, the mais component
a-Endorphin in Rat Pituitarq Gland
Vol . 25, No . 6, 1979
FIG. 1 Displacement curves of a-endorphin (a-EP), y-endorphin (y-EP), d-eacbrphin ( d-EP), ß-endorphin (ß-EP) and ß-lipotropia (ß-LPH) in radioimmunoassay of a-endorphin. The staadard curve of a-endorphin shows that this assay systeem is suitable for measuring a-endorphin, 80 to 1800 pg/tube in amount. y-EP, 6 -EP, ß-EP, and ß-LPH cross react lean than 1 ~, on a molar basis when assessed by 80 ~, displaoameot of tracer binding to the antibody. ah-ACTS, ß-M~i, Meth-enke~ia ~~b_~ a~ ~b-min (Leu6-Enk) failed to diaplaoe ~I-aendorphin from its antibody.
Ok
0
PI N
PI N
u~anl~
FFG. 2 EBect of extraction p>
Pergaawn Preas
PRESENCE OF IMMUNOREACTIVE a-ENDORPHIN IN RAT P1TUIi'ARY C3LAND Jtitnichi Fukata, Yoehikatsu Nalrai and Hiroo Imura god Division, Department of hiternal Medicine, Kyoto University Faculty of Medicine, Kyoto 808, Japan. (Received in final fors July 2, 1979)
Utilizing radioimmunoassay for a-e~ndoiphin, we attempted to demnonetrate immunoreactive a-endorphin in acid eatracte of pare distalis a~ oomhined pans intermedia and parr nervosa aS the rat P 9.Blaad attar chromatography on Sephadex Gr2ä ßLipotropin, ~-endorphin a~ y-endorphin orere not converted i~o aemdorphia during the extraction and gel chromatographic procedures . Ca~noemtrations of immunoreactlve a-endorphin determined after gel chromatography of e~dr8ots from pare distalir and combined pare intsrmedis and parr nervosa were L 1±0, 8 and 130±1? ng/mg ~ throe (mean±SE), respectively" ~ Serial dilution of these e~dracts gave parallel lines to the standard curve a~f synthetic a-endbrphin, but not to that of y-endorphin or 6-endorphin. These results suggest the existence of immunoreactive a-endorphin indistinguishable in molecular size from syntheüc a-endorphin in the rat pituitary gland. Boon after the etiuoture of enkephalins wan identified from porcine brain (1), three opioid peptides, a-endorphin, ß-endorphin and Y-esdoiphtn were isolated from pituitary tirsue or the bypothalanno-pituitary comple~c (2, 3) " Recently, the oonoentra bona of ~-endorphin (4, 6) and Y-endorphin (8) in rat pituitary glatds have been measured by radioimmumorrray and the existence of a-endorphin, another opioid peptide, in the rat pituitary glad has bees suggested by Hlocmn et al who used the ianmtmooytofluoreroenoe technique (?). To our lmowledge, however, tbere has been no report caacerntag the concentration of a-endorphin in rat pituitary gland. The present rtud9 was designed ~ demonstrate the exirtemoe of a-endorphin and to measure its concentration in rat Pituitary glaada utilizitg radioimm~moassgy for aendorphin. Materials sad Meth Extraction of a-Endorphin. Three male Wistar rats weighing 400-860 g were decapitated. Pare distalis and combined pare intermedia and pans nervosa of the pituitary gland were carefully separated, weighed and immersed in 2 ml of chilled 0. 2N HCi. All the procedures were completed within one or two minutes. The tisane was boiled for 10 min at 96'C, then immediately cooled and homogenized üpr 90 sec with a Polyfron homogenizer. After centrifhgation at 10, OOOsg in a reirlgerated centxlfhge 0024-3205/79/060541-06$02 .00/0 Copyright (c) 1979 Perganon Press Ltd
54 2
a-Endorphin in Rat Pituitary Gland
Vol . 25, No . 6, 1979
for 30 min, the supernatant was collected ànd lyophilized. The lyophiilized materials were the reconstituted in 0. 06M phosphate buffer (pH 7 . 4) containing 0. 5 ~ human serum albumin, 500 KIU/ml Trasylol and 0. 4 ~ meroaptoethaaol (standard diluent). Recovery of immunorea+ctive a-endorphin during this extraction procedure, as monitored by the addition of 80 ng syntheric a-endorphin to 100 mg of rat liver rissue, was 53 ~. Gel Exclusion Chromatography. A 0. 2 ml aliquot of the reconsrituted solution or a syntheric a-emlorphin was applied on a 0. 7 x 57 om column of 8ephadex G-25 equilibrated and eluted with the standard diluent at 4'C . The flow rate was 1. 5 mI/h and the fraction volume was 0. 74 ml . The a-endorphin content of each fraction was 125I_ measured by radioimmunoassay . Blue dextrsa was used as a marker for Vo, ßh-endorphin (Li) for ß-endorphin sad 12bI for salt peak. Syntheric a-endorphin was used as a marker for a-endorphin, because 125I_a-endorphin eluted at a position slightly behind synthetic a-endorphin . Radioimmunoassay . Radioimmunoassay of a-endorphin was performed using antibody to synthetic a-endorphin (kindly provided by Dr . R. (~uillemin) at the final concentration of 1 :21, 000 . Synthetic a-endorphin was used ae a reference standard and for labeling . a-Endorphia was iodinated with Na 125I using the Chloramine T method of Hunter aml Greenwood (8) . Purlücation of 125I_a-eadorphin was carried out by chromatography oa 3ephadex G-25. The specinc acrivriy of 1251-a-endorphin ranged from 180 to 180 uCi/Rg. Radioimmunoassay was performed by the double antibody immunopreoipitarion technique, utilizing anti-rabbit y-globulin aarisera as the second antibody. The minimal detectable quality of a-endorphin was 20 pg" Ira- and interassay coefficie~s of variation were 3. 4 % and 8. 3 ~, respectively . This antiserum showed weak cross-reactivity with ß-lipotropin, ß-endorphin, y-endorphin and d -endorphin . Hut, Met~enkephalin, heu 5-~kephalin, ß-MSH and human ACTH failed to displace 125 1-a-endorphin from the antiserum eveâ when quantities as large as 10 ng were added (Fig. 1) . Radioimmumassay of ACTH was performed with the antiserum provided by the National Pituitary Agency (9) . Stability of B-endorphin, v-~dorohin and 8-lipotropin during extraction procedure . To exclude the possibility of the conversion of ß-endorphin into a-endorphin in the process of extraction, 2. 4 l+g of sy~heric ß-endorphin was added to combined pans intermedia and pars nervosa of rat pituitaries immediately after separation, boiled and homogenized in 2 ml of chilled 0 . 2N HCI, ~ then extracted and reconsrituted in standard diluent. The preparation was then subjected to radioimmunoasasy for aeadorphin and ACTH. There was no conversion of ß-endorphin into a-endorphin during the extracrion procedure, 88 the ratios of a-endorphin to ACTH concentration from the pituitary extract with or without addition of 2. 4 ug of ß-endorphin were identical (Fig. 2) . In the same way, there was ao apparent conversion of ß-lipotropin . or y-endorphin into a-endorphin . Results The elution profiles of para distalis and combined pars intermedia and pars nervosa of rat pituitary gland are shown in Fig. 3. Ia the extract of pars distalis, two peaks with a-endorphin-like immunoreactivity were found; the first peak eluted in a poeirion compatible with 1251-ß_endorphin and the 2nd peak emerged at the elurioa position of synthetic a-endorphin (designated as immunoreactive a-endorphin hereafter) . Ia extract of combined para intermedia and pars nervosa, the mais component
a-Endorphin in Rat Pituitarq Gland
Vol . 25, No . 6, 1979
FIG. 1 Displacement curves of a-endorphin (a-EP), y-endorphin (y-EP), d-eacbrphin ( d-EP), ß-endorphin (ß-EP) and ß-lipotropia (ß-LPH) in radioimmunoassay of a-endorphin. The staadard curve of a-endorphin shows that this assay systeem is suitable for measuring a-endorphin, 80 to 1800 pg/tube in amount. y-EP, 6 -EP, ß-EP, and ß-LPH cross react lean than 1 ~, on a molar basis when assessed by 80 ~, displaoameot of tracer binding to the antibody. ah-ACTS, ß-M~i, Meth-enke~ia ~~b_~ a~ ~b-min (Leu6-Enk) failed to diaplaoe ~I-aendorphin from its antibody.
Ok
0
PI N
PI N
u~anl~
FFG. 2 EBect of extraction p>
