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      Synthesis of LH-RH by Rat Hypothalamic Tissue in vitro: I. Use of a Specific Antibody to LH-RH for Immunoprecipitation



      S. Karger AG

      LH-RH, Synthesis, Immunoprecipitation

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          Following the procedure of Jeffcoate et al. [1974] we have successfully obtained a specific antiserum to LH-RH in rabbits, and are utilizing our antibody to study the synthesis of LH-RH by rat hypothalamic tissues in vitro. Our antibody, used at a dilution of 1:20,000, binds 37% of added 125I-labelled LH-RH, and cross-reacts minimally with thyrotropin releasing factor (TRF; 4.0%), somatotropin release-inhibiting factor (SRIF; 0.035%), and melanocyte inhibiting factor (MIF; 0.009%). The antiserum recovers LH-RH added to charcoal-stripped rat serum quantitatively. Serial dilutions of rat hypothalamic extract and varying aliquots of normal rat serum are parallel to the standard curve (synthetic LH-RH: 0–100 pg/tube). Hypothalamic tissue of 4 male rats, bounded by the optic chiasm, mammillary bodies and hypothalamic fissures to a depth of 2–3 mm, was pooled, minced and incubated for 1–3 h in 2 ml of Eagle’s Minimum Essential Medium in an atmosphere of 95% O<sub>2</sub>/5% CO<sub>2</sub> with 10 µCi of <sup>3</sup>H-glycine. Reactions were stopped by the addition of 1 ml 0.1 n HC1. The tissue and medium were homogenized together and boiled for 3 min. Aliquots from the incubates were neutralized, LH-RH levels measured by radioimmunoassay (RIA), and 3H-glycine incorporation determined by immunoprecipitation, using our antibody. <sup>3</sup>H-glycine incorporation into presumptive LH-RH increased linearly over the 3 h period. Aliquots of the 3 h incubates were chromatographed on Sephadex G-25 columns (1 × 10 cm), using 0.01 m acetic acid for elution; 1 ml fractions were collected. Synthetic LH-RH was chromatographed in a similar manner, and its elution profile determined by UV absorbance at 280 mm. Aliquots of each fraction of the eluted material from the 3 h incubates were counted in a scintillation counter to determine the elution pattern of the labelled material. Additionally, aliquots from the 1 ml fractions were neutralized and used for immunoprecipitation. Coincident peaks of <sup>3</sup>H-label, immunoprecipitable <sup>3</sup>H-labelled presumptive LH-RH, and synthetic LH-RH were observed. These results lend strong evidence to support our conclusion that the technique of immunoprecipitation is an efficacious approach to the study of the synthesis of LH-RH by rat hypothalamic tissue in vitro.

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          Author and article information

          S. Karger AG
          25 March 2008
          : 21
          : 2
          : 111-119
          Department of Reproductive Medicine and Biology, University of Texas Medical School at Houston, Houston, Tex.
          122517 Neuroendocrinology 1976;21:111–119
          © 1976 S. Karger AG, Basel

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          Page count
          Pages: 9


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