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Human big gastrin N-terminal fragment immunoreactivity in tissue and blood
S123
HUMAN BIG GASTRIN N-TERMINAL FRAGMENT IMMUNOREACTIVITY IN TISSUE AND BLOOD N. Yanaihara, S. Mihara, F. Shimizu, K. Nagai, K. Iguchi, M. Sato, M. Sakagami, C. Yanaihara, T. lwanaga*, Y. Kusumoto* and T. Fujita* Laboratory of Bioorganic Chemistry, Shizuoka College of Pharmacy, Shizuoka, and aDept of Anatomy, Niigata Univ School of Medicine, Niigata, Japan The newly synthesized hG34 N-terminal pentadecapeptide [hG34(i-15)] conclusively assessed immunologically usefulness of radioimmunoassay with an extremely N-terminal specific anti-hG34(l-15) serum R-2702 which had been developed with the synthetic peptide having the original sequence. In~nunohistochemical staining of human antrum and duodenum using antiserum R-2702 demonstrated hG34 Nterminal immunoreactivity in the G cells. The immunoreactivities in human stomach and duodenum measured by the R-2702 assay system were shown by gel filtration on Sephadex G50 (superfine) to consist of major components of molecular weights identical with or lower than synthetic hG34(i-15). Rat stomach extracts showed the elution profile of gel filtration on Sephadex G50 (superfine) comprising two major components which seemed to correspond to hG34 and the N-terminal fragment. The dose-response curves of the components were however not parallel to the standard. In ten healthy male volunteers (ages 23-25) given at noon hospital test meal (protein 41.5 g, carbohydrate 37.5 g, fat 11.6 g, energy 437 cal), plasma level of hG34 N-terminal immunoreactivity, 16 + 6 pg hG34(i15) equivalent/ml, was gradually increased and reached the maximum value, 70 + 18 pg/ml, 45 min after the meal. The immunoreactivity was then decreased and the level after 90 min was 48 + 14 pg.ml. A major part of the increased hG34 N-terminal immunoreactivity in-the plasma was interpreted by components having Kav-- 0.73 on Sephadex G50 (superfine) gel filtration and a component co-eluting with [125-I]-synthetic G34(I-15) marker (Ka. ~ 0.58) was rather minor. The present results support usefulness of the radio~mmunoassay using antiserum R-2702 for measurement of blood level of hG34 and its N-terminal fragment immunoreactivities. SYNTHETIC STUDY ON PORCINE GASTRIN RELEASING PEPTIDE N. Yanaihara, T. Mochizuki, C. Yanaihara, V. Mutt* and T. McDonald** Laboratory of Bioorganic Chemistry, Shizuoka College of Pharmacy, Shizuoka, Japan, *Dept of Biochemistry II, Karolinska Institute, Stockholm, Sweden, and **Dept of Medicine, Univ of Western Ontario, London, Canada Synthesis of the non-antral gastric heptacosapeptide amide was carried out by azide fragment condensation method in solution. The entire sequence of the peptide was constructed using four protected peptide fragments: Z-AIa-Pro-VaI-SerVaI-GIy-GIy-NHNH2, Boc-Gly-Thr-Val-Leu-Ala-Lys(Tos)-Met-NHNH2, Boc-Tyr-Pro-ArgGIy-Asn-His-NHNH 2 and Boc-Trp-Ala-Val-Gly-His-Leu-Met-NH2. After successive azide condensation of these protected peptides starting from the C-terminal 2127 fragment, the ensuing protected heptacosapeptide amide was treated with sodium in liquid ammonia. The deprotected peptide amide was purified by gel filtration on Sephadex G25 followed by repeated ion-exchange chromatography on CM-cellulose (CM52). The synthetic heptacosapeptide amide contained the expected amino acids in the following ratios: Asp 1.09,Thr 1.05,Ser 1.01,Gly 5.22, Ala 3.21,Val 3.77,Met 1.99,Leu 1.99,Tyr 0.98,Trp 0.79,Lys 1.00,His 1.78,Arg 0.73,Pro 1.90. Isoelectrofocusing of the peptide (200 ~g) between pH 3.5 and i0 showed a single band in the region of about pH 8.5. Although HPLC analysis indicated some heterogeneity of the synthetic preparation, it evidently contained a major component which appeared to be identical with the natural porcine gastrin releasing peptide. The synthetic heptacosapeptide amide exhibited substantial gastrin releasing peptide-like bioactivity in the guinea pig gall bladder contracting assay. The synthetic C-terminal 14-27 fragment contained the bioactivity, while preliminary analysis suggested the synthetic N-terminal 1-13 fragment had no activity. Extremely close structural similarities of the ~-terminal 18-27 sequence of the porcine gastrin releasing peptide to amphibian bombesin may explain the bioactivity exhibited by the C-terminal fragment.
HUMAN BIG GASTRIN N-TERMINAL FRAGMENT IMMUNOREACTIVITY IN TISSUE AND BLOOD N. Yanaihara, S. Mihara, F. Shimizu, K. Nagai, K. Iguchi, M. Sato, M. Sakagami, C. Yanaihara, T. lwanaga*, Y. Kusumoto* and T. Fujita* Laboratory of Bioorganic Chemistry, Shizuoka College of Pharmacy, Shizuoka, and aDept of Anatomy, Niigata Univ School of Medicine, Niigata, Japan The newly synthesized hG34 N-terminal pentadecapeptide [hG34(i-15)] conclusively assessed immunologically usefulness of radioimmunoassay with an extremely N-terminal specific anti-hG34(l-15) serum R-2702 which had been developed with the synthetic peptide having the original sequence. In~nunohistochemical staining of human antrum and duodenum using antiserum R-2702 demonstrated hG34 Nterminal immunoreactivity in the G cells. The immunoreactivities in human stomach and duodenum measured by the R-2702 assay system were shown by gel filtration on Sephadex G50 (superfine) to consist of major components of molecular weights identical with or lower than synthetic hG34(i-15). Rat stomach extracts showed the elution profile of gel filtration on Sephadex G50 (superfine) comprising two major components which seemed to correspond to hG34 and the N-terminal fragment. The dose-response curves of the components were however not parallel to the standard. In ten healthy male volunteers (ages 23-25) given at noon hospital test meal (protein 41.5 g, carbohydrate 37.5 g, fat 11.6 g, energy 437 cal), plasma level of hG34 N-terminal immunoreactivity, 16 + 6 pg hG34(i15) equivalent/ml, was gradually increased and reached the maximum value, 70 + 18 pg/ml, 45 min after the meal. The immunoreactivity was then decreased and the level after 90 min was 48 + 14 pg.ml. A major part of the increased hG34 N-terminal immunoreactivity in-the plasma was interpreted by components having Kav-- 0.73 on Sephadex G50 (superfine) gel filtration and a component co-eluting with [125-I]-synthetic G34(I-15) marker (Ka. ~ 0.58) was rather minor. The present results support usefulness of the radio~mmunoassay using antiserum R-2702 for measurement of blood level of hG34 and its N-terminal fragment immunoreactivities. SYNTHETIC STUDY ON PORCINE GASTRIN RELEASING PEPTIDE N. Yanaihara, T. Mochizuki, C. Yanaihara, V. Mutt* and T. McDonald** Laboratory of Bioorganic Chemistry, Shizuoka College of Pharmacy, Shizuoka, Japan, *Dept of Biochemistry II, Karolinska Institute, Stockholm, Sweden, and **Dept of Medicine, Univ of Western Ontario, London, Canada Synthesis of the non-antral gastric heptacosapeptide amide was carried out by azide fragment condensation method in solution. The entire sequence of the peptide was constructed using four protected peptide fragments: Z-AIa-Pro-VaI-SerVaI-GIy-GIy-NHNH2, Boc-Gly-Thr-Val-Leu-Ala-Lys(Tos)-Met-NHNH2, Boc-Tyr-Pro-ArgGIy-Asn-His-NHNH 2 and Boc-Trp-Ala-Val-Gly-His-Leu-Met-NH2. After successive azide condensation of these protected peptides starting from the C-terminal 2127 fragment, the ensuing protected heptacosapeptide amide was treated with sodium in liquid ammonia. The deprotected peptide amide was purified by gel filtration on Sephadex G25 followed by repeated ion-exchange chromatography on CM-cellulose (CM52). The synthetic heptacosapeptide amide contained the expected amino acids in the following ratios: Asp 1.09,Thr 1.05,Ser 1.01,Gly 5.22, Ala 3.21,Val 3.77,Met 1.99,Leu 1.99,Tyr 0.98,Trp 0.79,Lys 1.00,His 1.78,Arg 0.73,Pro 1.90. Isoelectrofocusing of the peptide (200 ~g) between pH 3.5 and i0 showed a single band in the region of about pH 8.5. Although HPLC analysis indicated some heterogeneity of the synthetic preparation, it evidently contained a major component which appeared to be identical with the natural porcine gastrin releasing peptide. The synthetic heptacosapeptide amide exhibited substantial gastrin releasing peptide-like bioactivity in the guinea pig gall bladder contracting assay. The synthetic C-terminal 14-27 fragment contained the bioactivity, while preliminary analysis suggested the synthetic N-terminal 1-13 fragment had no activity. Extremely close structural similarities of the ~-terminal 18-27 sequence of the porcine gastrin releasing peptide to amphibian bombesin may explain the bioactivity exhibited by the C-terminal fragment.