0
views
0
recommends
+1 Recommend
1 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found

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

      ,

      Neuroendocrinology

      S. Karger AG

      LH-RH, Synthesis, Immunoprecipitation

      Read this article at

      ScienceOpenPublisherPubMed
      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          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.

          Related collections

          Author and article information

          Journal
          NEN
          Neuroendocrinology
          10.1159/issn.0028-3835
          Neuroendocrinology
          S. Karger AG
          0028-3835
          1423-0194
          1976
          1976
          25 March 2008
          : 21
          : 2
          : 111-119
          Affiliations
          Department of Reproductive Medicine and Biology, University of Texas Medical School at Houston, Houston, Tex.
          Article
          122517 Neuroendocrinology 1976;21:111–119
          10.1159/000122517
          796750
          © 1976 S. Karger AG, Basel

          Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

          Page count
          Pages: 9
          Categories
          Paper

          Comments

          Comment on this article