Life Scieaces Vol . 22, pp . Printed in the U.S .A .
2189-2196
Pergamon Preea
RADIOIMMUNOASSAY FOR ß-ENDORPHIN : PRESENCE OF IMMUNOREACTIVE "BIG-BIG" ß-ENDORPHIN ("BIG" ßLIPOTROPIN) IN HUMAN AND RAT PITUITARIES Hiroki Yoshimi, Shigeru Matsukura, Satoru Sueoka, Massaki Fukase, Makoto Yokota, Yukio Hirata and Hiroo Imura Third Division, Department of Medicine, Kobe University School of Medicine, Ikuta-ku, Robe 650, Japan . (Received in final form May 5, 1978)
Summary A sensitive and specific radioimmunoassay for ßendorphin has been developed with an antiserum obtained in a rabbit immunized with ß-endorphin contained in crude porcine ACTH preparations . The minimal detectable quantity was 5 pg . The antiserum used reacted slightly with ovine ß-lipotropin (5 .5 $), but showed negligible cross-reactivity with other fragments of ßUsing this radioimmunolipotropin, a-MSH and ACTH . assay we have observed the presence of "big-big" ßendorphin ("big" ß-lipotropin) with apparent molecular weights of 37,000 and 31,000 in human and rat pituitaries respectively, in addition to ß-lipotropin and ßendorphin, by Sephadex gel-chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis . Among the various fragments of ß-lipotropin (ß-LPH), ßendorphin, recently isolated from camel (1), ovine (2), bovine (3) and human (4) pituitaries and subsequently synthesized (5), is found to be the most potent endogenous morphinomimetic peptide by bioassay (6, 7) and opiate receptor binding assay (8) . However, these assays are not sensitive and specific enough to investigate the physiological role of B-endorphin . Recently, several more sensitive radioimmunoassays for ß-endorphin were reported (9-13) . We have also developed a highly sensitive and specific radioimmunoassay for ß-endorphin and observed the presence of "big-big" ß-endorphin . ("big" ß-LPH), ß-LPH and ß-endorphin in human and rat pituitary extracts by Sephadex gel-chromatography and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis . Materials and Methods The antiserum (R974) against B-endorphin was produced in a 0300-9653/78/0626-2189$02 .00/0 Copyright Q 1978 Pergamon Preaa
2190
Large Molecular Endorphine
Vol . 22, No . 24, 1978
rabbit immunized with crude porcine ACTH preparations (Organon) emulsified in complete Freund's adjuvant (Difco) . Highly purified human g-endorphin, which had been kindly supplied by Dr . C . H . Li, was labelled with Na i25I using chloramine T according to the method of Hunter and Greenwood (14) . The purification of labelled ß-endorphin was performed on a carboxymethyl cellulose column (CM52 Whatman) : The specific activity of 1251-ß-endorphin ranged from 140 to 180 uCi/yg . Preparation of Samples : Acetone-dried human pituitary powder was mixed in 4 ml 1 M acetic acid, boiled for a minute and stirred overnight at 4 C . The mixture was then centrifuged at 2500 g for 10 min and the supernatant was collected and lyophilized . Apart of the lyophilized extract was reconstituted in 2 ml 0 .1 M acetic acid . After insoluble material was removed by centrifugation, the supernatant was applied to Sephadex G-100 column (1 .5 X 90 cm) . The column was equilibrated and eluted with 0 .1 M acetic acid at a flow rate of 14 ml/hr . Each 3 ml fraction was collected and stored at -20 C until assayed . Rat pituitaries and crude porcine ACTH preparations (Organon) were also extracted and processed as described above . Polyacrylamide Gel Electrophoresis : SDS polyacrylamide gel electrophoresis (15 $ acrylamide, 0 .4 $ N, N'-methylenebisacrylamide) was prepared according to a modification of the method of Ring and Laemmli (15) . The radioimmunoassay for ß-endorphin was unaffected by the concentrations of SDS as high as 0 .05 mg/0 .5 ml, so it was possible to carry out radioimmunoassay for ß-endorphin after gel electrophoresis . After electrophoresis gels were cut into slices of 2 mm length and eluted into 0 .5 ml of 0 .05 $ SDS and 5 mM NaHC03 for 8 hr at 37 C, the elutee was removed and assayed . Radioimmunoassay Procedure : 0 .05 M phosphate buffer, pH 7 .4 containing 0 .5 $ human serum albumin (Armour), 500 RIU /ml of Trasylol (Bayer) and 0 .5 8 2-mercaptoethanol (Nakarai, Kyoto) was used to prepare all solutions and as a diluent . 0 .1 ml anti-ßendorphin serum (diluted to 1 :8,000), 0 .1 ml standard or unknown ~~mple, 0 .2 ml phosphate buffer and approximately 5,000 cpm of I-ß-endorphin in 0 .1 ml phosphate buffer were mixed and incubated for 72 hr at 4 C . The separation of antibody bound from free labelled hormone was done by adsorption to 50 mg of talcum . ACTH was also determined by specific radioimmunoassay using synthetic human ACTH (Shionogi, Osaka) as tracer and standard and anti-ACTH serum provided by the National Institute of Arthritis, Metabolism, and Digestive Diseases, NIH . Results A typical standard curve with ß-endorphin is shown in Fig . 1. Significant inhibition of the binding of 125I~ -endorphin to antibody is evident with as little as 5 pg of g-endorphin . This antiserum (R974) showed slight cross-reactivity with highly purified ovine B-LPH (5 .5 8) but synthetic Mets-enkephalin, Leusenkephalin, a-endorphin, human ß-MSH, a-MSH and human ACTH failed to displace 125 1-ß-endorphin from its antibody, even when quantities as much as 10 ng were added . Intra- and inter-assay coefficients of variation were 6 .2 $ and 8 .6 8, respectively .
Vol . 22, No . 24, 1978
Large Molecular Endorphine
2191
FIG. 1 Fig . 1 . The standard curve of radioimmunoassay for ßendorphin (EP) and cross-reaction with fragmenta of 9LPH and Leur-enkephalin, a-MSH, human ß-MSH and human ACTH . This antiserum (R974) to ß-endorphin shows slight reaction with ßs-LPH (5 .5 8) but negligible cross-reaction with other peptides . Elution patterns on Sephadex G-100 gel chromatography of extracts of crude porcine ACTH preparations (Organon) and of human and rat pituitary were shown in Fig . 2 and Fig . 3, respec tively . Fractionation of extract of crude porcine ACTH preparations (Organon) resulted in only one peak with ß-endorphin immu-
4~0 2 ~ n u FIG . 2 Fig . 2 . Elution profile of extract of crude porcine ACTH preparations (Organon) on Sephadex G-100 (1 .5 x 90 cm) gel filtration . See Materials and Methods for detailed procedures .
2192
Larges Molecular Bndorphins
FRACTION
Vol. 22, No . 24, 1978
MlwIBER
FIG . 3 Fig . 3 . Elution profiles of human (upper) and rat (lower) pituitary extracts on Sephadex G-100 (1 .5 X 90 cm) gel filtrations . See Materials and Methods for detailed procedures . noreactivity, eluted in a position where 125 1-ß_endorphin emerged (Fig . 2) . However, fractionations of extracts of human and rat pituitaries revealed apparently three peaks with ß-endorphin immunoreactivity (Fig . 3) ; the first peak eluted near the void volume, the second in a position compatible with 125 I_ßs-LPH and the third in a position of 125 1-ß-endorphin . Eluates collected near the void volume from fractionations of human and rat pituitary èxtracts (Fig . 3) were lyophylized and analyzed by SDS-polyacrylamide gel electrophoresis . After elec trophoresis of human and rat pituitary extracts three peaks of ßendorphin immunoreactivity were resolved (Fig . 4) ; the first peak of B-endorphin immunoreactivity ["big-big" ß-endorphin ("big" ßLPH)] with molecular weight of 37,000 in human pituitary extract or 31,000 in rat pituitary extract (as described in the legend of Fig . 4), the second and the third peaks compatible with ß-LPH and 9-endorphin, respectively . Simultaneously ACTH antigenicity of each gel slice elutee was determined by radioimmunoassay and four peaks of ACTH immunoreactivity were resolved in human and rat pituitary extracts, respectively . Only the first peak of ACTH immunoreactivity coincided with each peak of B-endorphin immunoreactivity with the largest molecular weight in human pituitary extract or in rat pituitary extract . Discussion We have successfully obtained the antiserum to B-endorphin
Vol. 22, No . 24, 1978
Large Molecular Endorphine
2193
r 5
4 6 DMane~(em)
"
10
FIG . 4 Fig . 4 . Sodium dodecyl sulfate polyacrylamide gel electrophoresis of eluates collected near the void volume from Sephadex gel fractionations of human (upper) and rat (lower) pituitary extracts . The eluates were lyophilized, dissolved in 2 .2 $ sodium dodecyl sulfate, 5 .4 mM EDTA, 10 .9 $ glycerol, 0 .068 mM Tris HC1 buffer, pH 6 .8 and 5 .4 8 2-mercaptoethanol, and incubated in boiling water for 5 min . The samples were analyzed by gel electrophoresis as described under Materials and Methods . Ovalbumin (molecular weight 43,000), LDH (molecular weight 36,000) and chymotrypsinogen (molecular weight 25,700) had mobilities of 0 .19, 0 .28 and 0 .38, respectively . Calibration of the molecular weight range from 43,000 to 25,700 by these gels gave a straight line . in a rabbit immunized with B-endorphin contained in crude porcine ACTH preparations (Organon), which was confirmed by qelchromatographic study (as shown in Fig . 2) . Because of the negligible cross-reaction with a-endorphin and Mets-enkephalin, this antiserum (R974) was considered to be directed to 17-31 amino acid sequence of the ß-endorphin molecule, which is not shared with .these two morphinomimetic peptides . Since the amino acid sequence of sheep B-endorphin was found to be same as that of porcine ß-endorphin except for the 23th amino acid (16), the antiserum used here was considered to react also with sheep ßendorphin and B-LPH . Using this radioimmunoassay we have demonstrated the pre-
2194
Large Molecular Endorphins
Vol . 22, No . 24, 1978
since of "big-big" ß-endorphin ("big" B-LPH) with molecular weight of 37,000 in human pituitary extract or 31,000 in rat pituitary extract, in addition to B-LPH and ß-endorphin, by Sephadex gel-chromatography and SDS-polyacrylamide gel electrophoresis . It was unexpected that eluates collected near the void volume from Sephadex G-100 gel fractionations were found to contain g-LPH and B-endorphin besides "big-big" ß-endorphin ("big" ß-LPH) by gel electrophoresis . This result may indicate the size-heterogeneity of peptides fractionated on gel chromatography or the partial conversion of big to smaller molecules during gel electrophoresis . ß-endorphin immunoreactivity which moved faster than the third peak (Fig, 4) was considered to be artifacts due to interference in subsequent radioimmunoassays, since high concentrations of SDS contained in 2 .2 $ SDS-iris HC1 buffer used for appling the sample were found to comigrate faster with bromphenol blue during gel electrophoresis and were eluted . Very recently, Mains et al . (11) reported the presence of the common precursor to ACTH and ß-endorphin, with molecular weight of 31,000, in culture medium from the AtT-20 mouse pituitary tumor cells using sequential double-antibody immunoprecipitation technique . Roberts and Herbert (17) have also reported that the cellfree product of AtT-20 mouse cells may be the common precursor to both peptides . It is likely that "big-big" ß-endorphin ("big" ßLPH) with apparent molecular weights of 37,000 and 31,000 in human and rat pituitary extracts, respectively, is identical to the common precursor proposed by them (11,17), since in the present study ACTH antigenicity was also detected in these fractions . However, to prove this point we should need sequential immunoprecipitation study and should, moreover, clarify the enzymatic conversion of "big-big" ß-endorphin to B-LPH and/or B-endorphin . We were unable to find "big-big" ß-endorphin and ß-LPH in crude porcine ACTH preparations (Organon) . The reason for this is not apparent . The absence of "big-big" ß-endorphin from the crude porcine ACTH preparations could well be due to the method of preparation of crude ACTH by oxycellulose adsorption, since the basicity of "big-big" B-endorphin is unknown . There is also the possibility of complete degradation of parent B-LPH before the purification of porcine pituitaries . Li and Chung (1) also reported that they could not isolate ß-LPH from camel pituitaries and speculated the possibility that g-LPH had been completely degraded during the prolonged transportation of camel pituitaries to laboratory . Acknowledgement We wish to thank Dr . C .H . Li for human ß-endorphin . References C . H . LI and D . CHUNG, Proc . Natl . Acad . Sci . USA _73 11451148 (1976) . M . CHRETIEN, S . BENJANNET, N . DRAGON, N . G . SEIDAH, and M . LIS, Biochem. Biophys . Res_ ._ Commun . 72 472-478 (1976) . C . H . LI, . TAN, H G, B oc em . Biophys . Res . Commun . 7 7 1088-1093 (1977) . C . H . LI, D . CHUNG, B . A . DONEEN, Biochem . Biophys . Res . Commun . 7 2 1542-1547 (1976) .
Vol . 22, No . 24, 1978 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 .
Large Molecular Endorphine
2195
C . H . LI, S . LEMAIRE, D . YAMASHIRO, B . A . DONEEN, Biochem . Biophys . Res . Commun . 7 1 19-25 (1976) . L . GRAF, J . I . SZEKELY,A . Z . RONAI, Z . DUNAI-KOVACS, S . BAJUSZ, Nature 263 240-241 (1976) . H . H . LOH, L . FTSENG, . E . WEI, C . H . LI, Proc . Natl . Acad . Sci . USA 73 2895-2897 (1976) . L . H . LAZARUS, N . LING, R . GUILLEMIN, Proc . Natl . Acnd . Sci . US A 73 2156-2158 (1976) . .BLOOM, E . BATTENBERG, J . ROSSIER, N . LING, J . LEPPALUOTO, F T . M. VARGO, R . GUILLEMIN, Life Sci . 20 43-48 (19I7) . R . GUILLEMIN, N . LING, T . VARGÔ ioB c~m . Biophys . Res . Commun . 7 7 361-366 (1977) . R . E . MAINS, B . A . EIPPER, N . LING, Proc . Natl . Acnd . Sci . US A 74 3014-3018 (1977) . .T.KRIEGER, A . LIOTTA, T . SUDA, M . PALKOVITS, M . J . D BROWNSTEIN, Bîochem . Biophys . Res . Commun . 7 6 930-936 (1977) . C . H . LI, J . RAO, B . A . DONEEN, D . YAMASHIRÔ, Biochem . Bioh s . Res . Commun . 75 576-580 (1977) . F . C . G ENWOOD, W . M. HUNTER, and J . C . GLOVER, Bîochem . J . 89 114-123 (1963) . J . KING, and U . K . T.ARMMT.j, J, Mol . Biol . 62 465-477 (1971) . C . H . LI and D . CHUNG, Nature - 4 Ti976) . J . L . ROBERTS and E . HERBERT, Proc . Natl . Acnd . Scî . USA _74 5300-5304 (1977) .
2189-2196
Pergamon Preea
RADIOIMMUNOASSAY FOR ß-ENDORPHIN : PRESENCE OF IMMUNOREACTIVE "BIG-BIG" ß-ENDORPHIN ("BIG" ßLIPOTROPIN) IN HUMAN AND RAT PITUITARIES Hiroki Yoshimi, Shigeru Matsukura, Satoru Sueoka, Massaki Fukase, Makoto Yokota, Yukio Hirata and Hiroo Imura Third Division, Department of Medicine, Kobe University School of Medicine, Ikuta-ku, Robe 650, Japan . (Received in final form May 5, 1978)
Summary A sensitive and specific radioimmunoassay for ßendorphin has been developed with an antiserum obtained in a rabbit immunized with ß-endorphin contained in crude porcine ACTH preparations . The minimal detectable quantity was 5 pg . The antiserum used reacted slightly with ovine ß-lipotropin (5 .5 $), but showed negligible cross-reactivity with other fragments of ßUsing this radioimmunolipotropin, a-MSH and ACTH . assay we have observed the presence of "big-big" ßendorphin ("big" ß-lipotropin) with apparent molecular weights of 37,000 and 31,000 in human and rat pituitaries respectively, in addition to ß-lipotropin and ßendorphin, by Sephadex gel-chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis . Among the various fragments of ß-lipotropin (ß-LPH), ßendorphin, recently isolated from camel (1), ovine (2), bovine (3) and human (4) pituitaries and subsequently synthesized (5), is found to be the most potent endogenous morphinomimetic peptide by bioassay (6, 7) and opiate receptor binding assay (8) . However, these assays are not sensitive and specific enough to investigate the physiological role of B-endorphin . Recently, several more sensitive radioimmunoassays for ß-endorphin were reported (9-13) . We have also developed a highly sensitive and specific radioimmunoassay for ß-endorphin and observed the presence of "big-big" ß-endorphin . ("big" ß-LPH), ß-LPH and ß-endorphin in human and rat pituitary extracts by Sephadex gel-chromatography and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis . Materials and Methods The antiserum (R974) against B-endorphin was produced in a 0300-9653/78/0626-2189$02 .00/0 Copyright Q 1978 Pergamon Preaa
2190
Large Molecular Endorphine
Vol . 22, No . 24, 1978
rabbit immunized with crude porcine ACTH preparations (Organon) emulsified in complete Freund's adjuvant (Difco) . Highly purified human g-endorphin, which had been kindly supplied by Dr . C . H . Li, was labelled with Na i25I using chloramine T according to the method of Hunter and Greenwood (14) . The purification of labelled ß-endorphin was performed on a carboxymethyl cellulose column (CM52 Whatman) : The specific activity of 1251-ß-endorphin ranged from 140 to 180 uCi/yg . Preparation of Samples : Acetone-dried human pituitary powder was mixed in 4 ml 1 M acetic acid, boiled for a minute and stirred overnight at 4 C . The mixture was then centrifuged at 2500 g for 10 min and the supernatant was collected and lyophilized . Apart of the lyophilized extract was reconstituted in 2 ml 0 .1 M acetic acid . After insoluble material was removed by centrifugation, the supernatant was applied to Sephadex G-100 column (1 .5 X 90 cm) . The column was equilibrated and eluted with 0 .1 M acetic acid at a flow rate of 14 ml/hr . Each 3 ml fraction was collected and stored at -20 C until assayed . Rat pituitaries and crude porcine ACTH preparations (Organon) were also extracted and processed as described above . Polyacrylamide Gel Electrophoresis : SDS polyacrylamide gel electrophoresis (15 $ acrylamide, 0 .4 $ N, N'-methylenebisacrylamide) was prepared according to a modification of the method of Ring and Laemmli (15) . The radioimmunoassay for ß-endorphin was unaffected by the concentrations of SDS as high as 0 .05 mg/0 .5 ml, so it was possible to carry out radioimmunoassay for ß-endorphin after gel electrophoresis . After electrophoresis gels were cut into slices of 2 mm length and eluted into 0 .5 ml of 0 .05 $ SDS and 5 mM NaHC03 for 8 hr at 37 C, the elutee was removed and assayed . Radioimmunoassay Procedure : 0 .05 M phosphate buffer, pH 7 .4 containing 0 .5 $ human serum albumin (Armour), 500 RIU /ml of Trasylol (Bayer) and 0 .5 8 2-mercaptoethanol (Nakarai, Kyoto) was used to prepare all solutions and as a diluent . 0 .1 ml anti-ßendorphin serum (diluted to 1 :8,000), 0 .1 ml standard or unknown ~~mple, 0 .2 ml phosphate buffer and approximately 5,000 cpm of I-ß-endorphin in 0 .1 ml phosphate buffer were mixed and incubated for 72 hr at 4 C . The separation of antibody bound from free labelled hormone was done by adsorption to 50 mg of talcum . ACTH was also determined by specific radioimmunoassay using synthetic human ACTH (Shionogi, Osaka) as tracer and standard and anti-ACTH serum provided by the National Institute of Arthritis, Metabolism, and Digestive Diseases, NIH . Results A typical standard curve with ß-endorphin is shown in Fig . 1. Significant inhibition of the binding of 125I~ -endorphin to antibody is evident with as little as 5 pg of g-endorphin . This antiserum (R974) showed slight cross-reactivity with highly purified ovine B-LPH (5 .5 8) but synthetic Mets-enkephalin, Leusenkephalin, a-endorphin, human ß-MSH, a-MSH and human ACTH failed to displace 125 1-ß-endorphin from its antibody, even when quantities as much as 10 ng were added . Intra- and inter-assay coefficients of variation were 6 .2 $ and 8 .6 8, respectively .
Vol . 22, No . 24, 1978
Large Molecular Endorphine
2191
FIG. 1 Fig . 1 . The standard curve of radioimmunoassay for ßendorphin (EP) and cross-reaction with fragmenta of 9LPH and Leur-enkephalin, a-MSH, human ß-MSH and human ACTH . This antiserum (R974) to ß-endorphin shows slight reaction with ßs-LPH (5 .5 8) but negligible cross-reaction with other peptides . Elution patterns on Sephadex G-100 gel chromatography of extracts of crude porcine ACTH preparations (Organon) and of human and rat pituitary were shown in Fig . 2 and Fig . 3, respec tively . Fractionation of extract of crude porcine ACTH preparations (Organon) resulted in only one peak with ß-endorphin immu-
4~0 2 ~ n u FIG . 2 Fig . 2 . Elution profile of extract of crude porcine ACTH preparations (Organon) on Sephadex G-100 (1 .5 x 90 cm) gel filtration . See Materials and Methods for detailed procedures .
2192
Larges Molecular Bndorphins
FRACTION
Vol. 22, No . 24, 1978
MlwIBER
FIG . 3 Fig . 3 . Elution profiles of human (upper) and rat (lower) pituitary extracts on Sephadex G-100 (1 .5 X 90 cm) gel filtrations . See Materials and Methods for detailed procedures . noreactivity, eluted in a position where 125 1-ß_endorphin emerged (Fig . 2) . However, fractionations of extracts of human and rat pituitaries revealed apparently three peaks with ß-endorphin immunoreactivity (Fig . 3) ; the first peak eluted near the void volume, the second in a position compatible with 125 I_ßs-LPH and the third in a position of 125 1-ß-endorphin . Eluates collected near the void volume from fractionations of human and rat pituitary èxtracts (Fig . 3) were lyophylized and analyzed by SDS-polyacrylamide gel electrophoresis . After elec trophoresis of human and rat pituitary extracts three peaks of ßendorphin immunoreactivity were resolved (Fig . 4) ; the first peak of B-endorphin immunoreactivity ["big-big" ß-endorphin ("big" ßLPH)] with molecular weight of 37,000 in human pituitary extract or 31,000 in rat pituitary extract (as described in the legend of Fig . 4), the second and the third peaks compatible with ß-LPH and 9-endorphin, respectively . Simultaneously ACTH antigenicity of each gel slice elutee was determined by radioimmunoassay and four peaks of ACTH immunoreactivity were resolved in human and rat pituitary extracts, respectively . Only the first peak of ACTH immunoreactivity coincided with each peak of B-endorphin immunoreactivity with the largest molecular weight in human pituitary extract or in rat pituitary extract . Discussion We have successfully obtained the antiserum to B-endorphin
Vol. 22, No . 24, 1978
Large Molecular Endorphine
2193
r 5
4 6 DMane~(em)
"
10
FIG . 4 Fig . 4 . Sodium dodecyl sulfate polyacrylamide gel electrophoresis of eluates collected near the void volume from Sephadex gel fractionations of human (upper) and rat (lower) pituitary extracts . The eluates were lyophilized, dissolved in 2 .2 $ sodium dodecyl sulfate, 5 .4 mM EDTA, 10 .9 $ glycerol, 0 .068 mM Tris HC1 buffer, pH 6 .8 and 5 .4 8 2-mercaptoethanol, and incubated in boiling water for 5 min . The samples were analyzed by gel electrophoresis as described under Materials and Methods . Ovalbumin (molecular weight 43,000), LDH (molecular weight 36,000) and chymotrypsinogen (molecular weight 25,700) had mobilities of 0 .19, 0 .28 and 0 .38, respectively . Calibration of the molecular weight range from 43,000 to 25,700 by these gels gave a straight line . in a rabbit immunized with B-endorphin contained in crude porcine ACTH preparations (Organon), which was confirmed by qelchromatographic study (as shown in Fig . 2) . Because of the negligible cross-reaction with a-endorphin and Mets-enkephalin, this antiserum (R974) was considered to be directed to 17-31 amino acid sequence of the ß-endorphin molecule, which is not shared with .these two morphinomimetic peptides . Since the amino acid sequence of sheep B-endorphin was found to be same as that of porcine ß-endorphin except for the 23th amino acid (16), the antiserum used here was considered to react also with sheep ßendorphin and B-LPH . Using this radioimmunoassay we have demonstrated the pre-
2194
Large Molecular Endorphins
Vol . 22, No . 24, 1978
since of "big-big" ß-endorphin ("big" B-LPH) with molecular weight of 37,000 in human pituitary extract or 31,000 in rat pituitary extract, in addition to B-LPH and ß-endorphin, by Sephadex gel-chromatography and SDS-polyacrylamide gel electrophoresis . It was unexpected that eluates collected near the void volume from Sephadex G-100 gel fractionations were found to contain g-LPH and B-endorphin besides "big-big" ß-endorphin ("big" ß-LPH) by gel electrophoresis . This result may indicate the size-heterogeneity of peptides fractionated on gel chromatography or the partial conversion of big to smaller molecules during gel electrophoresis . ß-endorphin immunoreactivity which moved faster than the third peak (Fig, 4) was considered to be artifacts due to interference in subsequent radioimmunoassays, since high concentrations of SDS contained in 2 .2 $ SDS-iris HC1 buffer used for appling the sample were found to comigrate faster with bromphenol blue during gel electrophoresis and were eluted . Very recently, Mains et al . (11) reported the presence of the common precursor to ACTH and ß-endorphin, with molecular weight of 31,000, in culture medium from the AtT-20 mouse pituitary tumor cells using sequential double-antibody immunoprecipitation technique . Roberts and Herbert (17) have also reported that the cellfree product of AtT-20 mouse cells may be the common precursor to both peptides . It is likely that "big-big" ß-endorphin ("big" ßLPH) with apparent molecular weights of 37,000 and 31,000 in human and rat pituitary extracts, respectively, is identical to the common precursor proposed by them (11,17), since in the present study ACTH antigenicity was also detected in these fractions . However, to prove this point we should need sequential immunoprecipitation study and should, moreover, clarify the enzymatic conversion of "big-big" ß-endorphin to B-LPH and/or B-endorphin . We were unable to find "big-big" ß-endorphin and ß-LPH in crude porcine ACTH preparations (Organon) . The reason for this is not apparent . The absence of "big-big" ß-endorphin from the crude porcine ACTH preparations could well be due to the method of preparation of crude ACTH by oxycellulose adsorption, since the basicity of "big-big" B-endorphin is unknown . There is also the possibility of complete degradation of parent B-LPH before the purification of porcine pituitaries . Li and Chung (1) also reported that they could not isolate ß-LPH from camel pituitaries and speculated the possibility that g-LPH had been completely degraded during the prolonged transportation of camel pituitaries to laboratory . Acknowledgement We wish to thank Dr . C .H . Li for human ß-endorphin . References C . H . LI and D . CHUNG, Proc . Natl . Acad . Sci . USA _73 11451148 (1976) . M . CHRETIEN, S . BENJANNET, N . DRAGON, N . G . SEIDAH, and M . LIS, Biochem. Biophys . Res_ ._ Commun . 72 472-478 (1976) . C . H . LI, . TAN, H G, B oc em . Biophys . Res . Commun . 7 7 1088-1093 (1977) . C . H . LI, D . CHUNG, B . A . DONEEN, Biochem . Biophys . Res . Commun . 7 2 1542-1547 (1976) .
Vol . 22, No . 24, 1978 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 .
Large Molecular Endorphine
2195
C . H . LI, S . LEMAIRE, D . YAMASHIRO, B . A . DONEEN, Biochem . Biophys . Res . Commun . 7 1 19-25 (1976) . L . GRAF, J . I . SZEKELY,A . Z . RONAI, Z . DUNAI-KOVACS, S . BAJUSZ, Nature 263 240-241 (1976) . H . H . LOH, L . FTSENG, . E . WEI, C . H . LI, Proc . Natl . Acad . Sci . USA 73 2895-2897 (1976) . L . H . LAZARUS, N . LING, R . GUILLEMIN, Proc . Natl . Acnd . Sci . US A 73 2156-2158 (1976) . .BLOOM, E . BATTENBERG, J . ROSSIER, N . LING, J . LEPPALUOTO, F T . M. VARGO, R . GUILLEMIN, Life Sci . 20 43-48 (19I7) . R . GUILLEMIN, N . LING, T . VARGÔ ioB c~m . Biophys . Res . Commun . 7 7 361-366 (1977) . R . E . MAINS, B . A . EIPPER, N . LING, Proc . Natl . Acnd . Sci . US A 74 3014-3018 (1977) . .T.KRIEGER, A . LIOTTA, T . SUDA, M . PALKOVITS, M . J . D BROWNSTEIN, Bîochem . Biophys . Res . Commun . 7 6 930-936 (1977) . C . H . LI, J . RAO, B . A . DONEEN, D . YAMASHIRÔ, Biochem . Bioh s . Res . Commun . 75 576-580 (1977) . F . C . G ENWOOD, W . M. HUNTER, and J . C . GLOVER, Bîochem . J . 89 114-123 (1963) . J . KING, and U . K . T.ARMMT.j, J, Mol . Biol . 62 465-477 (1971) . C . H . LI and D . CHUNG, Nature - 4 Ti976) . J . L . ROBERTS and E . HERBERT, Proc . Natl . Acnd . Scî . USA _74 5300-5304 (1977) .
