0021-972X/79/4804-0559$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1979 by the Endocrine Society
Vol. 48, No. 4 Printed in U.S.A.
Analysis of Human Pituitary and Tumor Adrenocorticotropin Using Isoelectric Focusing* NOBUYOSHI TANAKA, KAORU ABE, SUMIKO MIYAKAWA, SHUMPEI OHNAMI, MASAHIRO TANAKA, AND TOSHIYUKI TAKEUCHI Endocrinology and Biochemistry Divisions, National Cancer Center Research Institute, Tokyo, Japan
ABSTRACT. Heterogeneity of immunoreactive ACTH was investigated in extracts of three human pituitary glands, three tumors obtained from patients with the ectopic ACTH syndrome, and a porcine pituitary gland using gel filtration and isoelectric focusing. A single gel filtration fraction of immunoreactive ACTH was applied to isoelectric focusing and pis were measured using RIAs specific for the amino-terminal portion (N-ACTH) and for the carboxyl-terminal (C-ACTH) of ACTH. The little ACTH peak of the human pituitary extract separated on gel filtration was found to contain four components with different pis on isoelectric focusing. The distribution of these peaks was almost identical in each pituitary extract, and the major peak in each case showed a pi value of 8.0, a value similar to that of synthetic human ACTH. The little ACTH
I
T HAS been demonstrated that ACTH has size heterogeneity when extracts of human pituitary glands, ectopic ACTH-producing tumors, or plasma of patients with elevated ACTH levels are subjected to gel filtration, even though they show a dose response parallel to the standard in the RIA (1-3). A single fraction of immunoreactive ACTH on gel filtration, however, does not necessarily represent a homogenous component of immunoreactive ACTH, because separation of peptide hormones on gel filtration, in general, is not sharp. Immunoreactive ACTH has been extensively studied by use of sodium dodecyl sulfate polyacrylamide gel electrophoresis; this procedure has revealed the multiple forms of immunoreactive ACTH with molecular weights of 31,000, 23,000, 13,000, and 4,500 (4). The size heterogeneity, however, cannot be determined using gel filtration or sodium dodecyl sulfate polyacrylamide gel electrophoresis if the difference in molecular sizes of immunoreactive Received August 7,1978. Address requests for reprints to: Kaoru Abe, M.D., Endocrinology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104, Japan. * This work was supported by grants for cancer research and for specific disease from the Ministry of Health and Welfare, by grants-inaid for scientific research from the Ministry of Education, Science, and Culture and by a grant from the Society for Promotion of Cancer Research, Japan.
peak of the tumor extract was composed of four or five peaks with different pi values. Each peak of the tumor extracts corresponded to some peak found in the pituitary extracts, and there was no peak specific for tumor. The C-fragment peaks in the tumor extracts and in the middle and posterior lobe extract of porcine pituitary, corresponding to corticotropin-like intermediate lobe peptide, showed a single peak with a pi of 5.2 and were different from the acidic forms of immunoreactive ACTH found in the little ACTH peak. These data indicate that isoelectric focusing is useful for examining microheterogeneity of immunoreactive ACTH with a similar molecular size when employed in conjunction with gel filtration. (J Clin Endocrinol Metab 48: 559, 1979)
ACTHs is due to a small number of amino acid residues, but ACTHs with small size difference may show ionic charge heterogeneity if a few amino acids lacking or surplus are of acidic or basic ionic charge. In this paper, we have applied isoelectric focusing to examine a single fraction of immunoreactive ACTH obtained by gel filtration. In general, isoelectric focusing is superior to electrophoresis for separation of proteins or polypeptides, although the principle of both techniques is based on ionic charge. The data obtained indicate that isoelectric focusing can be useful for further characterizing the immunoreactive ACTH peaks from gel filtration and may provide further information concerning the degradation of immunoreactive ACTH in vivo. Materials and Methods Synthetic human ACTH, used for iodination and assay standard, and synthetic human /?-MSH (y8-hMSH) were supplied from the National Pituitary Agency, NIH (Bethesda, MD). Synthetic human ACTH used for immunization was obtained from the Takeda Chemical Industries, Ltd. (Osaka, Japan). Synthetic materials such as a-ACTH110, a-ACTH"'24, aACTH25-39 a . A C T H i-24 a n d a_MSH were supplied from CibaGeigy, Ltd. (Basel, Switzerland). Highly purified porcine /?MSH was furnished by Dr. A. B. Lerner, Yale University (New Haven, CT); ovine /Mipotropic hormone (/?0-LPH) was provided by Dr. C. H. Li (San Francisco, CA). 559
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560
TANAKA ET AL.
Antisera were prepared in rabbits or guinea pigs immunized with synthetic human ACTH emulsified with complete Freund's adjuvant. Two antisera were selected according to the cross-reaction with ACTH fragments when [125I]human ACTH was used as a tracer. One antiserum raised in a rabbit reacted with a-ACTH1"39, a-ACTH1-24, and a-ACTH11-24 with a molar ratio of 1:1:0.25, but did not react with a-ACTH1-10, aACTH25"39, /MiMSH, porcine /3-MSH, or a-MSH in concentrations up to 1000 ng/tube. This antiserum was used to assay the amino-terminal portion of ACTH (N-assay). The other antiserum raised in a guinea pig reacted with a-ACTH139 and aACTH25"39 with a molar ratio of 1:1, but did not react with aACTH1"24, a-ACTH1"10, a-ACTH11-24, a-MSH, 0-hMSH, or fipMSH in concentrations up to 1000 ng/tube. This antiserum was used to assay the carboxyl-terminal portion of ACTH (Cassay). The RIA procedures of ACTH and fi-MSU were described previously (5, 6). Double antibody separation of bound from free hormones was used instead of paper chromatoelectrophoresis. The displacement of more than 2 SD of the trace bound to total hormones was always observed when 15 and 20 pg ACTH were added to the incubation tube for N- and C-assays, respectively. The inter- and intraassay coefficients of variation were 10% and 5% in N-assay and 12% and 6% in C-assay, respectively. Three human pituitary glands were obtained at autopsy from cancer patients without endocrine disease. Three tumors were obtained at the time of operation from patients with the ectopic ACTH syndrome; these consisted of two thymic carcinoids and a medullary carcinoma of the thyroid. The metastatic lung carcinoid tissue dissected from the liver, in which ACTH had not been detected, was used for determining the recovery rates of ACTH extraction and for the control study. A porcine pituitary gland was obtained from a nearby slaughterhouse. Immediately after removal, the tissues were stored at —20 C until extraction. Extraction was performed by methods reported previously, with some modifications (7). Frozen tissue was cut into small pieces and homogenized in a glass homogenizer with cold water (5 ml/g) for less than 1 min. The homogenization was repeated twice. The homogenate was transferred into 10 ml boiling water and kept at 100 C for 5 min. After addition of 1 ml 5 M acetic acid in an ice bath, the supernatant was collected after centrifugation at 1500 X g for 30 min at 4 C. Reextraction was performed by mixing the residue well with 10 ml 0.5 M acetic acid. The combined supernatant was then collected by centrifugation at 20,000 X g for 60 min at 4 C, lyophilized, and kept at -10 C in a desiccator until assayed. When 10, 50, and 250 ng synthetic human ACTH were added to the homogenates of liver metastatic tissue from the lung carcinoid, the recoveries were 46 ± 6%, 59 ± 4%, and 63 ± 9% (mean ± SD), respectively. For gel filtration, the lyophilized material was dissolved in 1.0 M acetic acid, stirred at room temperature for 30 min, and centrifuged at 1500 X g for 30 min at 4 C. The supernatant was then applied to a Sephadex gel column. The protein content of extracts was 4-8 mg/ml, determined by the method of Lowry et al. (8). The tissue extract was applied to a Sephadex G-50 column equilibrated with 1 M acetic acid and eluted with the same solution. Fractions were lyophilized and stored at 4 C. When
J C E & M • 1979 Vol 48 • No 4
applied to RIA, the lyophilized materials were dissolved in 1 ml standard diluent of RIA. The column was calibrated with defined standard materials. These were [125I]bovine serum albumin (New England Nuclear, Boston, MA), /?0-LPH, human ACTH1"39, 0-hMSH, 0-pMSH, and [125I]NaI. The hormones used for markers were assayed by the respective RIA after being eluted with 1 M acetic acid. Fractions separated on Sephadex G-50 were applied to isoelectric focusing. Electrofocusing was performed using a 110-ml column (LKB 8100-10, LKB instruments, Stockholm, Sweden) and carrier ampholite, pH 3.5-10 (LKB instruments), by the method of Haglund (9). The density gradient was prepared from 0-50% (wt/vol) sucrose with 0.5-1.5% (wt/vol) carrier ampholite. Specimens were inserted into the column close to the middle layer. Isoelectric focusing was performed at 4 C for 48 h with a voltage of 400-900 V. A total of 80 fractions of 1.4 ml each were collected. As control, 1.5 jug synthetic human ACTH were subjected to isoelectric focusing. Also, 1.5 jug synthetic human ACTH were added to the homogenate of liver metastatic tissue from the lung carcinoid, extracted with the same procedure, and subjected to isoelectric focusing. Results Gel filtration patterns of three human pituitary extracts on Sephadex G-50 are illustrated in Fig. 1. Immunoreactive ACTHs determined by N-assay (iV-ACTH) and C-assay (C-ACTH) showed identical gel filtration patterns. The fraction of big ACTH appeared in the excluded volume and comprised 7-9% of the total ACTH immunoreactivity. Most of the immunoreactive ACTH was eluted in the position of synthetic human ACTH1"39. Gel filtration patterns of three tumor extracts are shown in Fig. 2. AT-ACTH showed two peaks corresponding to big ACTH (which represents little ACTH alone) and little ACTH which appeared in the position of human ACTH1"39. C-ACTH had three peaks which corresponded to big ACTH, little ACTH, and C-fragment, the latter with a molecular weight roughly corresponding to synthetic /MiMSH. The C-fragment peak was not well separated from the little ACTH peak in the cases of tumors 1 and 3. The ratio of big ACTH to total immunoreactive ACTH was quite variable. When compared in N-assay, it was found to be 8% in tumor 1, 52% in tumor 2, and 3% in tumor 3. The C-fragment peaks were observed only in the tumor extracts; most of immunoreactive ACTH in tumor extract 2 consisted of C-fragment. Gel filtration patterns of a porcine pituitary gland are shown in Fig. 3. In the anterior pituitary extract, NACTH and C-ACTH corresponded well, making a small peak of big ACTH and a large peak of little ACTH. In the middle and posterior pituitary extract, however, NACTH was observed only in the position of little ACTH, while C-ACTH appeared as a large peak in the position of the C-fragment.
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ISOELECTRIC ANALYSIS OF ACTH
Vo£o-LPH
h39
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FIG. 1. Gel filtration patterns of three human pituitary extracts on Sephadex G-50 column (1.6 X 60 cm) eluted with 1 M acetic acid. • • , Immunoreactive ACTH determined by N-assay; O- -O, Immunoreactive ACTH determined by C-assay. •—O, Values of N- and C-assays are identical. Vo, Excluded volume determined by [125I]albumin; Vt, total bed volume determined by [l25I]NaI. The hormones used for markers are shown at the top.
FIG. 2. Gel filtration patterns of three tumor extracts on a Sephadex G-50 column (1.6 x 60 cm) eluted with 1 M acetic acid. • — • , Immunoreactive ACTH determined by N-assay; O—O, immunoreactive ACTH determined by C-assay. •—O, Values of N- and C-assays are identical. Vo, Excluded volume determined by [125I]albumin; Vt, total bed volume determined by [l25I]NaI. The hormones used for markers are shown at the top.
When the little ACTH peaks of human pituitary extracts were subjected to isoelectric focusing, four fractions with pis of 4.5-6.0, 6.0-7.0, 7.5-8.5, and over 9.5 were observed, as shown in Fig. 4. The fraction of pi 7.58.5 was the major one and pi of the peak was 8.0. NACTH and C-ACTH also corresponded well. Isoelectric focusing of the little ACTH peaks of tumor extracts revealed four or five pi peaks as shown in Fig. 5. Each pi peak showed the better separation when compared with those observed in the pituitary extracts. In tumors 1 and 2, two peaks in the pi range of 4.5-6.0 and one peak each in the pi ranges of 6.0-7.0 and 7.5-8.5 were observed. In tumor 3, five peaks were observed; one peak each in the pi ranges of 4.5-6.0, 7.5-8.5, and 9.0-10.0, and two peaks in the pi range of 6.0-7.0. The major peak was
at pi 8.0 in tumors 2 and 3. There was no pi 9.5 peak in tumors 1 and 2, and no pi 6.2 peak in tumor 2. Each pi peak found in the tumor extracts, however, corresponded to some peak found in the pituitary extracts, and there was no peak specific for tumor ACTH. A/-ACTH and CACTH also corresponded well in these cases, except for a pi 5.2 peak in tumor 3, in which little ACTH and Cfragment did not separate well on gel filtration. Distribution of the C-fragment peaks of tumor extracts on isoelectric focusing is shown in Fig. 6. C-ACTH showed a remarkable peak at pi 5.2, with lesser peaks at pi 5.8 in tumor 1 and at pi 8.0 in tumor 3. N-ACTH did not show any significant peaks. Results of isoelectric focusing of the porcine pituitary extract are shown in Fig. 7. The little ACTH peak of the
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TANAKA ET AL.
562
Vo flo-LPH
l39
hACTH
flh-MSH
Vt
JCK&M • 1979 Vol 48 • No 4
30
20-
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30
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Number
FIG. 3. Gel filtration patterns of porcine pituitary gland extracts on Sephadex G-50 (1.0 x 40 cm) eluted with 1 M acetic acid. The extract of the anterior pituitary is shown in the upper panel, and that of the middle and posterior pituitaries are shown in the lower panel. • — • , lmmunoreactive ACTH determined by iV-assay; O- -O, immunoreactive ACTH determined by C-assay. •—O, Values of N- and C-assays are identical. Vo, Excluded volume determined by [125I]albumin; Vt, total bed volume determined by [125I]NaI. The hormones used for markers are shown at the top.
anterior pituitary extract showed a main peak of pi 8.0, with two other small peaks at pi 5.8 and 6.8. The Cfragment peak of the middle and posterior pituitary extract revealed a single pi 5.2 peak. In the control experiments, synthetic human ACTH, which was subjected to isoelectric focusing with and without the same extraction procedure, showed a single peak of pi 8.0 (Fig. 8).
Discussion The little ACTH peak of the human pituitary extract separated on gel filtration was found to contain four components with different pis on isoelectric focusing. The distribution of these peaks was almost identical in each pituitary extract, and the major peak in each case showed a pi value of 8.0. In cases of tumor extracts, the little ACTH peak was found to be composed of four or five peaks with different pi values. The peaks observed
pH FIG. 4. Isoelectric focusing of the little ACTH peak of human pituitary extracts. A part of the gel filtrate with the highest ACTH content was lyophilized and dissolved in standard RIA diluent. This was applied to a 110-ml column with carrier ampholite, pH 3.5-10. • — • , lmmunoreactive ACTH determined by iV-assay; O- -O, immunoreactive ACTH determined by C-assay. •—O, N- and C-assay values are identical.
in cases of tumor extracts were sharper when compared with those in the pituitary extracts. This may indicate that the tumor tissues obtained at surgery were fresher than the pituitary glands obtained at the time of autopsy. Each peak of the tumor extracts, however, corresponded to some peak found in the pituitary extracts, and there was no peak specific for tumor. We presumed that tumor extracts might have pi peaks different from those of the pituitary. On the contrary, however, the tumor extracts showed a distribution of pi peaks similar to the pituitary peaks. It is clear from the control experiments that these
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563
ISOELECTRIC ANALYSIS OF ACTH
60
I
40
II
20 I I
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Vol. 48, No. 4 Printed in U.S.A.
Analysis of Human Pituitary and Tumor Adrenocorticotropin Using Isoelectric Focusing* NOBUYOSHI TANAKA, KAORU ABE, SUMIKO MIYAKAWA, SHUMPEI OHNAMI, MASAHIRO TANAKA, AND TOSHIYUKI TAKEUCHI Endocrinology and Biochemistry Divisions, National Cancer Center Research Institute, Tokyo, Japan
ABSTRACT. Heterogeneity of immunoreactive ACTH was investigated in extracts of three human pituitary glands, three tumors obtained from patients with the ectopic ACTH syndrome, and a porcine pituitary gland using gel filtration and isoelectric focusing. A single gel filtration fraction of immunoreactive ACTH was applied to isoelectric focusing and pis were measured using RIAs specific for the amino-terminal portion (N-ACTH) and for the carboxyl-terminal (C-ACTH) of ACTH. The little ACTH peak of the human pituitary extract separated on gel filtration was found to contain four components with different pis on isoelectric focusing. The distribution of these peaks was almost identical in each pituitary extract, and the major peak in each case showed a pi value of 8.0, a value similar to that of synthetic human ACTH. The little ACTH
I
T HAS been demonstrated that ACTH has size heterogeneity when extracts of human pituitary glands, ectopic ACTH-producing tumors, or plasma of patients with elevated ACTH levels are subjected to gel filtration, even though they show a dose response parallel to the standard in the RIA (1-3). A single fraction of immunoreactive ACTH on gel filtration, however, does not necessarily represent a homogenous component of immunoreactive ACTH, because separation of peptide hormones on gel filtration, in general, is not sharp. Immunoreactive ACTH has been extensively studied by use of sodium dodecyl sulfate polyacrylamide gel electrophoresis; this procedure has revealed the multiple forms of immunoreactive ACTH with molecular weights of 31,000, 23,000, 13,000, and 4,500 (4). The size heterogeneity, however, cannot be determined using gel filtration or sodium dodecyl sulfate polyacrylamide gel electrophoresis if the difference in molecular sizes of immunoreactive Received August 7,1978. Address requests for reprints to: Kaoru Abe, M.D., Endocrinology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104, Japan. * This work was supported by grants for cancer research and for specific disease from the Ministry of Health and Welfare, by grants-inaid for scientific research from the Ministry of Education, Science, and Culture and by a grant from the Society for Promotion of Cancer Research, Japan.
peak of the tumor extract was composed of four or five peaks with different pi values. Each peak of the tumor extracts corresponded to some peak found in the pituitary extracts, and there was no peak specific for tumor. The C-fragment peaks in the tumor extracts and in the middle and posterior lobe extract of porcine pituitary, corresponding to corticotropin-like intermediate lobe peptide, showed a single peak with a pi of 5.2 and were different from the acidic forms of immunoreactive ACTH found in the little ACTH peak. These data indicate that isoelectric focusing is useful for examining microheterogeneity of immunoreactive ACTH with a similar molecular size when employed in conjunction with gel filtration. (J Clin Endocrinol Metab 48: 559, 1979)
ACTHs is due to a small number of amino acid residues, but ACTHs with small size difference may show ionic charge heterogeneity if a few amino acids lacking or surplus are of acidic or basic ionic charge. In this paper, we have applied isoelectric focusing to examine a single fraction of immunoreactive ACTH obtained by gel filtration. In general, isoelectric focusing is superior to electrophoresis for separation of proteins or polypeptides, although the principle of both techniques is based on ionic charge. The data obtained indicate that isoelectric focusing can be useful for further characterizing the immunoreactive ACTH peaks from gel filtration and may provide further information concerning the degradation of immunoreactive ACTH in vivo. Materials and Methods Synthetic human ACTH, used for iodination and assay standard, and synthetic human /?-MSH (y8-hMSH) were supplied from the National Pituitary Agency, NIH (Bethesda, MD). Synthetic human ACTH used for immunization was obtained from the Takeda Chemical Industries, Ltd. (Osaka, Japan). Synthetic materials such as a-ACTH110, a-ACTH"'24, aACTH25-39 a . A C T H i-24 a n d a_MSH were supplied from CibaGeigy, Ltd. (Basel, Switzerland). Highly purified porcine /?MSH was furnished by Dr. A. B. Lerner, Yale University (New Haven, CT); ovine /Mipotropic hormone (/?0-LPH) was provided by Dr. C. H. Li (San Francisco, CA). 559
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560
TANAKA ET AL.
Antisera were prepared in rabbits or guinea pigs immunized with synthetic human ACTH emulsified with complete Freund's adjuvant. Two antisera were selected according to the cross-reaction with ACTH fragments when [125I]human ACTH was used as a tracer. One antiserum raised in a rabbit reacted with a-ACTH1"39, a-ACTH1-24, and a-ACTH11-24 with a molar ratio of 1:1:0.25, but did not react with a-ACTH1-10, aACTH25"39, /MiMSH, porcine /3-MSH, or a-MSH in concentrations up to 1000 ng/tube. This antiserum was used to assay the amino-terminal portion of ACTH (N-assay). The other antiserum raised in a guinea pig reacted with a-ACTH139 and aACTH25"39 with a molar ratio of 1:1, but did not react with aACTH1"24, a-ACTH1"10, a-ACTH11-24, a-MSH, 0-hMSH, or fipMSH in concentrations up to 1000 ng/tube. This antiserum was used to assay the carboxyl-terminal portion of ACTH (Cassay). The RIA procedures of ACTH and fi-MSU were described previously (5, 6). Double antibody separation of bound from free hormones was used instead of paper chromatoelectrophoresis. The displacement of more than 2 SD of the trace bound to total hormones was always observed when 15 and 20 pg ACTH were added to the incubation tube for N- and C-assays, respectively. The inter- and intraassay coefficients of variation were 10% and 5% in N-assay and 12% and 6% in C-assay, respectively. Three human pituitary glands were obtained at autopsy from cancer patients without endocrine disease. Three tumors were obtained at the time of operation from patients with the ectopic ACTH syndrome; these consisted of two thymic carcinoids and a medullary carcinoma of the thyroid. The metastatic lung carcinoid tissue dissected from the liver, in which ACTH had not been detected, was used for determining the recovery rates of ACTH extraction and for the control study. A porcine pituitary gland was obtained from a nearby slaughterhouse. Immediately after removal, the tissues were stored at —20 C until extraction. Extraction was performed by methods reported previously, with some modifications (7). Frozen tissue was cut into small pieces and homogenized in a glass homogenizer with cold water (5 ml/g) for less than 1 min. The homogenization was repeated twice. The homogenate was transferred into 10 ml boiling water and kept at 100 C for 5 min. After addition of 1 ml 5 M acetic acid in an ice bath, the supernatant was collected after centrifugation at 1500 X g for 30 min at 4 C. Reextraction was performed by mixing the residue well with 10 ml 0.5 M acetic acid. The combined supernatant was then collected by centrifugation at 20,000 X g for 60 min at 4 C, lyophilized, and kept at -10 C in a desiccator until assayed. When 10, 50, and 250 ng synthetic human ACTH were added to the homogenates of liver metastatic tissue from the lung carcinoid, the recoveries were 46 ± 6%, 59 ± 4%, and 63 ± 9% (mean ± SD), respectively. For gel filtration, the lyophilized material was dissolved in 1.0 M acetic acid, stirred at room temperature for 30 min, and centrifuged at 1500 X g for 30 min at 4 C. The supernatant was then applied to a Sephadex gel column. The protein content of extracts was 4-8 mg/ml, determined by the method of Lowry et al. (8). The tissue extract was applied to a Sephadex G-50 column equilibrated with 1 M acetic acid and eluted with the same solution. Fractions were lyophilized and stored at 4 C. When
J C E & M • 1979 Vol 48 • No 4
applied to RIA, the lyophilized materials were dissolved in 1 ml standard diluent of RIA. The column was calibrated with defined standard materials. These were [125I]bovine serum albumin (New England Nuclear, Boston, MA), /?0-LPH, human ACTH1"39, 0-hMSH, 0-pMSH, and [125I]NaI. The hormones used for markers were assayed by the respective RIA after being eluted with 1 M acetic acid. Fractions separated on Sephadex G-50 were applied to isoelectric focusing. Electrofocusing was performed using a 110-ml column (LKB 8100-10, LKB instruments, Stockholm, Sweden) and carrier ampholite, pH 3.5-10 (LKB instruments), by the method of Haglund (9). The density gradient was prepared from 0-50% (wt/vol) sucrose with 0.5-1.5% (wt/vol) carrier ampholite. Specimens were inserted into the column close to the middle layer. Isoelectric focusing was performed at 4 C for 48 h with a voltage of 400-900 V. A total of 80 fractions of 1.4 ml each were collected. As control, 1.5 jug synthetic human ACTH were subjected to isoelectric focusing. Also, 1.5 jug synthetic human ACTH were added to the homogenate of liver metastatic tissue from the lung carcinoid, extracted with the same procedure, and subjected to isoelectric focusing. Results Gel filtration patterns of three human pituitary extracts on Sephadex G-50 are illustrated in Fig. 1. Immunoreactive ACTHs determined by N-assay (iV-ACTH) and C-assay (C-ACTH) showed identical gel filtration patterns. The fraction of big ACTH appeared in the excluded volume and comprised 7-9% of the total ACTH immunoreactivity. Most of the immunoreactive ACTH was eluted in the position of synthetic human ACTH1"39. Gel filtration patterns of three tumor extracts are shown in Fig. 2. AT-ACTH showed two peaks corresponding to big ACTH (which represents little ACTH alone) and little ACTH which appeared in the position of human ACTH1"39. C-ACTH had three peaks which corresponded to big ACTH, little ACTH, and C-fragment, the latter with a molecular weight roughly corresponding to synthetic /MiMSH. The C-fragment peak was not well separated from the little ACTH peak in the cases of tumors 1 and 3. The ratio of big ACTH to total immunoreactive ACTH was quite variable. When compared in N-assay, it was found to be 8% in tumor 1, 52% in tumor 2, and 3% in tumor 3. The C-fragment peaks were observed only in the tumor extracts; most of immunoreactive ACTH in tumor extract 2 consisted of C-fragment. Gel filtration patterns of a porcine pituitary gland are shown in Fig. 3. In the anterior pituitary extract, NACTH and C-ACTH corresponded well, making a small peak of big ACTH and a large peak of little ACTH. In the middle and posterior pituitary extract, however, NACTH was observed only in the position of little ACTH, while C-ACTH appeared as a large peak in the position of the C-fragment.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 06:34 For personal use only. No other uses without permission. . All rights reserved.
ISOELECTRIC ANALYSIS OF ACTH
Vo£o-LPH
h39
hACTH /3h-MSH
1 Vo /3o-LPH
vt
i
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561
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Number
30
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Vi.
40
50
60
Fraction Number
FIG. 1. Gel filtration patterns of three human pituitary extracts on Sephadex G-50 column (1.6 X 60 cm) eluted with 1 M acetic acid. • • , Immunoreactive ACTH determined by N-assay; O- -O, Immunoreactive ACTH determined by C-assay. •—O, Values of N- and C-assays are identical. Vo, Excluded volume determined by [125I]albumin; Vt, total bed volume determined by [l25I]NaI. The hormones used for markers are shown at the top.
FIG. 2. Gel filtration patterns of three tumor extracts on a Sephadex G-50 column (1.6 x 60 cm) eluted with 1 M acetic acid. • — • , Immunoreactive ACTH determined by N-assay; O—O, immunoreactive ACTH determined by C-assay. •—O, Values of N- and C-assays are identical. Vo, Excluded volume determined by [125I]albumin; Vt, total bed volume determined by [l25I]NaI. The hormones used for markers are shown at the top.
When the little ACTH peaks of human pituitary extracts were subjected to isoelectric focusing, four fractions with pis of 4.5-6.0, 6.0-7.0, 7.5-8.5, and over 9.5 were observed, as shown in Fig. 4. The fraction of pi 7.58.5 was the major one and pi of the peak was 8.0. NACTH and C-ACTH also corresponded well. Isoelectric focusing of the little ACTH peaks of tumor extracts revealed four or five pi peaks as shown in Fig. 5. Each pi peak showed the better separation when compared with those observed in the pituitary extracts. In tumors 1 and 2, two peaks in the pi range of 4.5-6.0 and one peak each in the pi ranges of 6.0-7.0 and 7.5-8.5 were observed. In tumor 3, five peaks were observed; one peak each in the pi ranges of 4.5-6.0, 7.5-8.5, and 9.0-10.0, and two peaks in the pi range of 6.0-7.0. The major peak was
at pi 8.0 in tumors 2 and 3. There was no pi 9.5 peak in tumors 1 and 2, and no pi 6.2 peak in tumor 2. Each pi peak found in the tumor extracts, however, corresponded to some peak found in the pituitary extracts, and there was no peak specific for tumor ACTH. A/-ACTH and CACTH also corresponded well in these cases, except for a pi 5.2 peak in tumor 3, in which little ACTH and Cfragment did not separate well on gel filtration. Distribution of the C-fragment peaks of tumor extracts on isoelectric focusing is shown in Fig. 6. C-ACTH showed a remarkable peak at pi 5.2, with lesser peaks at pi 5.8 in tumor 1 and at pi 8.0 in tumor 3. N-ACTH did not show any significant peaks. Results of isoelectric focusing of the porcine pituitary extract are shown in Fig. 7. The little ACTH peak of the
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TANAKA ET AL.
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FIG. 3. Gel filtration patterns of porcine pituitary gland extracts on Sephadex G-50 (1.0 x 40 cm) eluted with 1 M acetic acid. The extract of the anterior pituitary is shown in the upper panel, and that of the middle and posterior pituitaries are shown in the lower panel. • — • , lmmunoreactive ACTH determined by iV-assay; O- -O, immunoreactive ACTH determined by C-assay. •—O, Values of N- and C-assays are identical. Vo, Excluded volume determined by [125I]albumin; Vt, total bed volume determined by [125I]NaI. The hormones used for markers are shown at the top.
anterior pituitary extract showed a main peak of pi 8.0, with two other small peaks at pi 5.8 and 6.8. The Cfragment peak of the middle and posterior pituitary extract revealed a single pi 5.2 peak. In the control experiments, synthetic human ACTH, which was subjected to isoelectric focusing with and without the same extraction procedure, showed a single peak of pi 8.0 (Fig. 8).
Discussion The little ACTH peak of the human pituitary extract separated on gel filtration was found to contain four components with different pis on isoelectric focusing. The distribution of these peaks was almost identical in each pituitary extract, and the major peak in each case showed a pi value of 8.0. In cases of tumor extracts, the little ACTH peak was found to be composed of four or five peaks with different pi values. The peaks observed
pH FIG. 4. Isoelectric focusing of the little ACTH peak of human pituitary extracts. A part of the gel filtrate with the highest ACTH content was lyophilized and dissolved in standard RIA diluent. This was applied to a 110-ml column with carrier ampholite, pH 3.5-10. • — • , lmmunoreactive ACTH determined by iV-assay; O- -O, immunoreactive ACTH determined by C-assay. •—O, N- and C-assay values are identical.
in cases of tumor extracts were sharper when compared with those in the pituitary extracts. This may indicate that the tumor tissues obtained at surgery were fresher than the pituitary glands obtained at the time of autopsy. Each peak of the tumor extracts, however, corresponded to some peak found in the pituitary extracts, and there was no peak specific for tumor. We presumed that tumor extracts might have pi peaks different from those of the pituitary. On the contrary, however, the tumor extracts showed a distribution of pi peaks similar to the pituitary peaks. It is clear from the control experiments that these
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