Production of a recombinant newt growth hormone and its application for the development of a radioimmunoassay.
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 microl 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.
Yamamoto
K
K
School of Education, Waseda University, Nishiwaseda 1-6-1, Shinjuku-ku, Tokyo, 169-8050, Japan.
Takahashi
N
N
Nakai
T
T
Miura
S
S
Shioda
A
A
Iwata
T
T
Kouki
T
T
Kobayashi
T
T
Kikuyama
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D013006:Growth Hormone / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D010903:Pituitary Gland, Anterior / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D011863:Radioimmunoassay; D051381:Rats; D011994:Recombinant Proteins; D012455:Salamandridae / Q000235:genetics*; D012680:Sensitivity and Specificity; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology
D018076:DNA, Complementary; D011994:Recombinant Proteins; C404325:growth hormone, newt; D013973:Thyrotropin-Releasing Hormone; D013006:Growth Hormone