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Article: Production of recombinant goldfish prolactin and its applications in radioreceptor binding assay and radioimmunoassay

TitleProduction of recombinant goldfish prolactin and its applications in radioreceptor binding assay and radioimmunoassay
Authors
KeywordsGoldfish
Pituitary cells
Prolactin
Prolactin secretion
Radioimmunoassay
Receptor binding
Recombinant protein
Issue Date2002
PublisherAcademic Press. The Journal's web site is located at http://www.elsevier.com/locate/ygcen
Citation
General And Comparative Endocrinology, 2002, v. 126 n. 1, p. 75-89 How to Cite?
AbstractGoldfish prolactin cDNA was subcloned into a pRSET A vector and expressed in Escherichia coli. Recombinant goldfish prolactin was expressed mainly as insoluble inclusion bodies in the form of N-terminal 6x His-tagged fusion protein. This fusion protein was purified, refolded and 125I-labeled to generate a radioligand for receptor binding and validation of a radioimmunoassay for goldfish prolactin. Using goldfish gill membrane as the substrate for prolactin receptor binding, both recombinant and native forms of goldfish prolactin were effective in displacing the specific binding of the radioligand in a similar dose range, suggesting that the fusion protein was refolded properly and could be recognized by goldfish prolactin receptors. To quantify prolactin contents in biological samples from the goldfish, a radioimmunoassay using the 125I-labeled recombinant prolactin as a tracer was established. This assay was shown to be selective for goldfish prolactin without cross-reactivity with mammalian prolactin and pituitary hormones from other fish species (e.g., growth hormone and gonadotropin II). This newly validated assay system was used to investigate neuroendocrine and signal transduction mechanisms regulating prolactin release in the goldfish. In this case, the Ca2+ ionophore A23187 and protein kinase C activator TPA were effective in elevating basal levels of prolactin secretion in perifused goldfish pituitary cells. In parallel studies using a static incubation approach, somatostatin and dopamine, but not vasoactive intestinal polypeptide, were inhibitory to basal prolactin release in goldfish pituitary cells. These results suggest that somatostatin and dopamine may serve as negative regulators of basal prolactin secretion and that extracellular Ca2+ influx and protein kinase C activation may be important signaling events mediating prolactin release in the goldfish. ©2002 Elsevier Science (USA).
Persistent Identifierhttp://hdl.handle.net/10722/84635
ISSN
2015 Impact Factor: 2.667
2015 SCImago Journal Rankings: 1.245
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorWong, AOLen_HK
dc.contributor.authorCheung, HYSen_HK
dc.contributor.authorLee, EKYen_HK
dc.contributor.authorChan, KMen_HK
dc.contributor.authorCheng, CHKen_HK
dc.date.accessioned2010-09-06T08:55:21Z-
dc.date.available2010-09-06T08:55:21Z-
dc.date.issued2002en_HK
dc.identifier.citationGeneral And Comparative Endocrinology, 2002, v. 126 n. 1, p. 75-89en_HK
dc.identifier.issn0016-6480en_HK
dc.identifier.urihttp://hdl.handle.net/10722/84635-
dc.description.abstractGoldfish prolactin cDNA was subcloned into a pRSET A vector and expressed in Escherichia coli. Recombinant goldfish prolactin was expressed mainly as insoluble inclusion bodies in the form of N-terminal 6x His-tagged fusion protein. This fusion protein was purified, refolded and 125I-labeled to generate a radioligand for receptor binding and validation of a radioimmunoassay for goldfish prolactin. Using goldfish gill membrane as the substrate for prolactin receptor binding, both recombinant and native forms of goldfish prolactin were effective in displacing the specific binding of the radioligand in a similar dose range, suggesting that the fusion protein was refolded properly and could be recognized by goldfish prolactin receptors. To quantify prolactin contents in biological samples from the goldfish, a radioimmunoassay using the 125I-labeled recombinant prolactin as a tracer was established. This assay was shown to be selective for goldfish prolactin without cross-reactivity with mammalian prolactin and pituitary hormones from other fish species (e.g., growth hormone and gonadotropin II). This newly validated assay system was used to investigate neuroendocrine and signal transduction mechanisms regulating prolactin release in the goldfish. In this case, the Ca2+ ionophore A23187 and protein kinase C activator TPA were effective in elevating basal levels of prolactin secretion in perifused goldfish pituitary cells. In parallel studies using a static incubation approach, somatostatin and dopamine, but not vasoactive intestinal polypeptide, were inhibitory to basal prolactin release in goldfish pituitary cells. These results suggest that somatostatin and dopamine may serve as negative regulators of basal prolactin secretion and that extracellular Ca2+ influx and protein kinase C activation may be important signaling events mediating prolactin release in the goldfish. ©2002 Elsevier Science (USA).en_HK
dc.languageengen_HK
dc.publisherAcademic Press. The Journal's web site is located at http://www.elsevier.com/locate/ygcenen_HK
dc.relation.ispartofGeneral and Comparative Endocrinologyen_HK
dc.subjectGoldfishen_HK
dc.subjectPituitary cellsen_HK
dc.subjectProlactinen_HK
dc.subjectProlactin secretionen_HK
dc.subjectRadioimmunoassayen_HK
dc.subjectReceptor bindingen_HK
dc.subjectRecombinant proteinen_HK
dc.titleProduction of recombinant goldfish prolactin and its applications in radioreceptor binding assay and radioimmunoassayen_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=0016-6480&volume=126&spage=75&epage=89&date=2002&atitle=Production+of+Recombinant+Goldfish+Prolactin+and+Its+Applications+in+Radioreceptor+Binding+Assay+and+Radioimmunoassayen_HK
dc.identifier.emailWong, AOL: olwong@hkucc.hku.hken_HK
dc.identifier.authorityWong, AOL=rp00806en_HK
dc.description.naturelink_to_subscribed_fulltext-
dc.identifier.doi10.1006/gcen.2001.7771en_HK
dc.identifier.pmid11944969-
dc.identifier.scopuseid_2-s2.0-0036241466en_HK
dc.identifier.hkuros75796en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0036241466&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume126en_HK
dc.identifier.issue1en_HK
dc.identifier.spage75en_HK
dc.identifier.epage89en_HK
dc.identifier.isiWOS:000175237300012-
dc.publisher.placeUnited Statesen_HK
dc.identifier.scopusauthoridWong, AOL=7403147570en_HK
dc.identifier.scopusauthoridCheung, HYS=36899607300en_HK
dc.identifier.scopusauthoridLee, EKY=7406968652en_HK
dc.identifier.scopusauthoridChan, KM=7406032560en_HK
dc.identifier.scopusauthoridCheng, CHK=7404798014en_HK

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