Production of a recombinant newt growth hormone and its application for the development of a radioimmunoassay

Kazutoshi Yamamoto, Noriyuki Takahashi, Toshiki Nakai, Satoshi Miura, Akifumi Shioda, Takeo Iwata, Tom Kouki, Tetsuya Kobayashi, Sakae Kikuyama

    Research output: Contribution to journalArticle

    9 Citations (Scopus)

    Abstract

    Complementary DNA (cDNA) encoding newt (Cynops pyrrhogaster) growth hormone (nGH) was cloned from a cDNA library constructed from mRNAs of newt pituitary glands and was expressed in Escherichia coli. Based on Northern blot analysis using the cDNA as a probe, the nGH mRNA was estimated to be 940 bases in length. The recombinant nGH (nGHr) had a molecular mass of 22 kDa as determined by SDS-PAGE and possessed considerable bioactivity as determined in a Xenopus cartilage assay. Using the nGHr, we produced a polyclonal antibody against nGHr. Western blot analysis of newt anterior pituitary gland homogenates revealed that this antiserum specifically detected a single 22- kDa band, and histological studies of newt pituitary gland sections showed that the cells that reacted immunologically by the anti-nGHr antiserum corresponded to those stained by an antiserum against rat GH. A radioimmunoassay (RIA) that is specific and sensitive for nGH was developed, employing the antiserum thus produced. The sensitivity of the RIA was 57 ± 7 pg/100 μl assay buffer. Interassay and intraassay coefficients of variation were 1.22 and 2.70%, respectively. Serial dilutions of plasma and pituitary homogenate of C. pyrrhogaster yielded dose-response curves that were parallel to the standard curve. Plasma from hypophysectomized newts showed no cross- reactivity. Moreover, displacement curves obtained using pituitary homogenates of the sword-tailed newt (C. ensicauda) and the crested newt (Triturus carnifex) were also parallel to the standard curve. Mammalian and frog GHs and prolactins (PRLs), as well as newt PRL, showed no inhibition of binding, even at relatively high doses, in this RIA. The RIA was used to measure GH released from newt pituitaries in vitro. Enhancement of GH release by 10-7 M thyrotropin-releasing hormone was observed in cultures of newt pituitaries.

    Original languageEnglish
    Pages (from-to)103-116
    Number of pages14
    JournalGeneral and Comparative Endocrinology
    Volume117
    Issue number1
    DOIs
    Publication statusPublished - 2000 Jan

    ASJC Scopus subject areas

    • Endocrinology

    Fingerprint Dive into the research topics of 'Production of a recombinant newt growth hormone and its application for the development of a radioimmunoassay'. Together they form a unique fingerprint.

  • Cite this

    Yamamoto, K., Takahashi, N., Nakai, T., Miura, S., Shioda, A., Iwata, T., Kouki, T., Kobayashi, T., & Kikuyama, S. (2000). Production of a recombinant newt growth hormone and its application for the development of a radioimmunoassay. General and Comparative Endocrinology, 117(1), 103-116. https://doi.org/10.1006/gcen.1999.7387