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cerpta Medica, International Congress Series No 256: 94 Addison, G.M., c.N. Haies, J.S. Woodhead, J.LH. O'Riordan: (1972) Immunoradiometric assay of parathyroid hormone. J.EnJacobs, J. W., R. T. Sauer, H.D. Niall, H. T. Keutmann, J.L.H. docrinology 49: 521-530 (971) o 'R iordan , G.D. Aurbach and J. T. Potts: High sensitivity Arnaud, C.D., G. W. Sizemore, S.B. Oldham, J.A. Fischer, N-terminal sequenee analysis of human parathyroid horH.S. Tsao and E. T. Littledike: Human parathyroid hormone. Fed.Proc. 32: 648 (1973) (abstract) mone: glandular and secreted molecular species. Am.J. McJntosh, C.H.S., R.D. Hesch and J.S. Woodhead: The imMed. 50: 630-638 (1971) munoradiometric assay for human (1-34 )-parathyroid Arnaud, C. D.: Parathyroid hormone: Coming of age in c1inhormone. Second workshop on Vitamin D (1974) Wiesical medicine. Am.J.Med. 55 l577-581 (1973a) baden Arnaud, C.D.: Immunochemical heterogeneity of eireulating Mi/es, L.E.M. and C.H. Haies: The preparation and properties parathyroid hormone in man: Sequal to an original obof 131J-labelled antibodies to insulin. Biochem.J. 108: servation by Berson and Yalow. Mount Sinai J.Med. 40: 422-433 (1973b) 611-618 (1968) Readhead, c., G.M. Addison, c.N. Haies and H. Lehmann: Brewer, H.B., T. FairweIl, R. Ronan, G. W. Sizemore and Immunoradiometric and two-site assay of human folC.D. Arnaud: Human parathyroid hormone: Amino-acid Iicle-stimulating hormone. J.Endocrinol. 59: 313-323 sequence of the amino-terminal residues 1-34. Proc.Nat. (1973) Acad.Sci. USA 69: 3585-3588 (1972) Segre, G. V., J.F. Habener, D. Po weil, G. W. Treagar and J. T. Canterbury, J. and E. Reiss: Multiple immunoreactive molePotts: Parathyroid hormone in human plasma. J.Clin. eular forms of parathyroid hormone in human serum. l'lvest. 51: 3163-3168 (972) Proc.Soc.Exp.BioI.Med. 140-1393-1398 (1972) Silverman, R. and R. Yalow: Heterogeneity of parathyroid Goldsmith, R.S., J. Furszyfer, W.J. Johnson, A.E. Foumier, hormone. J.Clin.lnvest. 52: 1958-1971 (1973) G. W. Sizemore and C.D. Amaud: Etiology of hyperparathyroidism and bone disease during ehronic hemodialysis. Woodhead, 1.S., G.M. Addison and c.N. Haies: The immunoradiometrie assay and related techniques. Brit.Med.Bull. J.Clin.lnvest. 52: 173-180 (1973) 30: 44-49 (1974) Habener, J.F., B. Kemper, A. Rich and J. T. Potts: Biosynthesis of aprecursor to bovine parathyroid hormone. ExRequests for reprints should be addressed to: Dr. R.D. Heseh, Dept. Med., Univ. Hannover, D-3000 Hannover (Germany)

Short Communications Horm. Metab. Res. 7 (1975) 352-353

© Georg Thieme Verlag Stuttgart Human Growth Hormone Receptors in Human Circulating Lymphocytes*

R. Eshet l • S. Manheimer l • P. Chobsieng 2 and Z. Laron l I Institute of Pediatric and Adolescent Endocrinology, Beilinson Hospital, Petah Tikva, Department ot Physiology, Tel Aviv Univ. Med. School and 2Weitzman Institute of Sciences, Rehovot. Israel

Radioreceptor assays for HGH using different tissues have recently been developed. Tsuchima and Friesen (1973) described a radioreceptor assay using rabbit liver tissue homogenates. This assay did not discriminate between human, bovine, rat or rabbit growth hormone. Lesniak, Roth, Garden and Gavin (1973, 1974) described a specific radioreceptor assay for HGH using cultured human Iymphocytes. We hereby present a specific radioreceptor assay for HGH using fresh Iymphocytes from human peripheral blood. Material and Methods For iod in at ion the lactoperoxidase/glucose oxidase method of Miyachi, Chrambach, Mecklenburg and Lipsett (1973) was used. HGH Wilhelmi (NIH-GH HS/648E) was used for labelling as weil as for standard. The reaction mixture consisted of HGH, 2.5 lJg in 50 pl of 0.25 M phosphate buffer pH 6.0; 10 lJg glucose oxidase (Sigma) was added in 5 pl of the phosphate buffer; Lactoperoxidase (Sigma) in a concentration of 500 ng in 5 pl; glucose (11Jg) was added nine times in 5 pl phosphate buffer at 1 min intervals with stirring; the reaction was earried out at room temperature with 1 mCi 125-1. After iodination the hormone was purified on Sephadex G-50 according to Hunter and Greenwood (1962) using PBS 0.01 M, pH 7.4 for elution. Fractions of 1.0 ml were collected with 0.25% bovine serum albumin. The first peak containing 125-1 HGH at a specific activity of 80-150 f..LCi per lJg was used for the binding studies. The separation of the Iymphocytes from whole blood of adult donors was performed by the Ficoll density gradient centrifugation method of Boyum (1968) with modifications. The blood was diluted 1: 3 with PBS (phosphate buffered saline-Dulbecco-pH 7.2-7.4). The centrifuge tubes "'In partial fulfillment of the requirements for the Ph.D. degree of R.E. Supported by agrant from "The Israel Commission for Basie Research". Rec.: 20 Sept. 1974

Acc.: 20 May 1975

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containing 10 ml of a mixt ure of Ficoll 9% and diluted Isopaque 440 (2.4: 1.0) and 30 ml diluted blood were subjected to 1600 rpm for 30 min in a Hettich Roto silanta 111 centrifuge. Most of the Iymphocytes were found in the top band of the tube. They were resuspended and washed twice with PBS (Dulbecco) at 1000 rpm for 10 min and maintained in medium-199. Before use the cells were counted in a Neubauer chamber. The cells (lOxl0 6 in 0.48 ml medium-199 containing Penicillin 100 units/ml Streptomycin 100 J-Ig/ml) were added to llx70 mm plastic test tubes with the unlabelled hormone (10 IJI) for a preincubation of 30' at 30 0 C in a constant temperature water bath with virorous shaking. Then the labelIed hormone was added (100,000 cpm, 0.05 ng/l0 IJI) and the incubation carried out for another 90' at 300 C with shaking. After the incubation the tubes were centrifuged at 3000 rpm at 4 0 C for 5-10 min with one more wash in medium-199 and centrifugation. The cells were counted in a Packard Auto Gamma spectrometer. Results and Discussion

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We found that the 125-1 HGH bound by the circulating human Iymphocytes is displaced only by human growth hormone. The Figure shows the quantitative displacement of 125-1 HGH from the receptors on circulating Iymphocytes by unlabelled HGH. At a concentration of 0.1 ng/125-1 HGH per 0.5 ml of the circulating Iymphocytes OOxl0 6 cells) bound 34% of the total radioactivity. Unlabelled HGH at a concentration of 10 ng/ml displaced 20% of the bound radioactivity; 50 ng/ml displaced 32%; 500 ng/ml displaced 48.5% and 5000 ng/ml displaced 62.5%. Ovine GH and bovine GH inhibited the binding of 125-1 HGH only at concentrations over 50 J-Ig/ml. The lymphocytes preparat ion bound 34% of the total 125-1 insulin and 20-30% of the bound radioactivity was displaced by 10 ng/ml of standard insulin. We found no binding foe 131-1 Albumin. The measurement of the ability of a polypeptide hormone to compete for binding to a cellular receptor may give a estimate of its biological potency. This assay may be of importance in syndromes in which the presence of an immunoreactive but biologically inactive HGH (Laron, Pertzelan and Karp 1968) or adefeet of the receptor sites (Laron 1974) is suspected. In order to be useful in determining the presence of normal and abnormal struetured HGH the radioreeeptor assay of HGH must be sensitive and specific. The radioreeeptor assay using the rabbit liver cells (Tsuchima and Friesen 1973) does not meet the above criteria of specü·icity. The radioreeeptoe assay using cultured lymphocytes (Lesniak et al. 1973, 1974) meets the above eriteria, but does net permit direct testing of the receptors on the lymphocytes of a given patient. This limitation is overcome by the deseribed method using cireulating Iymphoeytes. This method is also simpler to perform than that using cultured Iymphoeytes.

Acknowledgements The authors are indebted to Dr. I. Koch, Dr. U. Zor and Ms. T. Baran, Weitzman Institute, and to Dr. I. Malehi, Beilinson Hospital. Referenees

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Boyum, A.: Scandinavian J.Cli.Lab.lnvest. Suppl. 97 (1968). - Hunter, W.M., F.C. Greenwood: Nature 194: 495 (1962). Laron, Z., A. Pertzelan, M. Karp: Israel J.Med.Sci. 4: 883 (1968). - Laron, Z.: Israel J.Med. Sei. 10: 1247 (1974). Lesniak, M.A., J. Roth, P. Gorden, J.R. Gavin 111: Nature New Biology 241: 20 (1973). - Lesniak, M.A., P. Gorden, J. Roth, IR. Gavin 111: J.BioI.Chem. 249: 1661 (1974). - Miyachi, J., A. Chrambach, R. Mecklenburg, M. Lipsett: Endocrinol. 92: 1725 (1973). - Tsuchima, T., H. Friesen: J.CIin.Endoerinol. 37: 334 (1973). Requests for reprints should be addressed to: Z. Laron, M.D., Beilinson Medical Center, Petah Tikva (Israel)

Horm. Metab. Res. 7 (1975) 353-354

© Georg Thieme Verlag Stuttgart Effect of Pig Neural Lobe Proteins on Rabbit Adipose Tissue M.J. Avery, W.B. Watkins and I.D_G_ Bevan ?ostgraduate School Obstet. and Gynaecology, University of Auekland, New Zealand There have been many reports indieating that extraets of pituitary glands possess lypolytic aetivity (Astwood, Barrett and Friesen 1961, Friesen, Barrett and Astwood 1962, Schleyer, Faulhaber, Voigt, Fehm and Pfeiffer 1971). Recently Rudman, Dei Rio, Garcia, Barnett, Ho ward, Walker and Moore (1970) isolated two lypolytie peptides from pig pituitaries. Since it Rec.: 2 Dee. 1974

Ace.: 21 Apr. 1975

Human growth hormone receptors in human circulating lymphocytes.

352 Short Communications cerpta Medica, International Congress Series No 256: 94 Addison, G.M., c.N. Haies, J.S. Woodhead, J.LH. O'Riordan: (1972) I...
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