Recombinant somatolactin as a stable and bioactive protein in a cell culture bioassay: development and validation of a sensitive and reproducible radioimmunoassay

Abstract

A recombinant somatolactin (SL) obtained by cloning and expression of sole SL cDNA was analyzed and used to develop a sensitive and specific RIA. In contrast to native proteins, which tend to dimerize and aggregate immediately after pituitary isolation, the majority of recombinant sole SL (rsSL) remained as a monomeric protein after long-term storage, as shown by size exclusion chromatography and Western blot. Using rsSL as a tracer and standard in the RIA, the minimum detectable dose and the midrange (ED 50 ) of the assay were 0·15 and 1·8-2·1 ng/ml respectively. Intra-and interassay coefficients of variation were 4·3% and 6·5% at ED 50 levels. Recombinant gilthead sea bream GH and recombinant trout GH did not show cross-reactivity, whereas a good parallelism between rsSL standard and serial dilutions of plasma and sole pituitary extracts was observed. In order to demonstrate some biological activity of rsSL, the ability of this recombinant product to prime gilthead sea bream phagocytes for in vitro enhancement of mitochondrial activity was examined by a chromogenic assay. A bell-shape dose-response curve was obtained with a maximum at 50 nM (1·2 µg/ml), similar to that reported previously for GH. Therefore, taking together all these data, it appears conclusive that rsSL is a long-term stable protein which retains, at least in part, biological activity, providing a useful tool to clarify the physiological role of fish SL.

Effects of constitutive expression of somatolactin alpha on skin pigmentation in medaka

“…1). Generally, hormones activate receptors in a dose-dependent manner, and a dose-response curve of SLa has been known to become bell shaped (Calduch-Giner et al, 1998); i.e., over-dosing inhibits activation (dimerization) of the receptor. We found that SLR (and also GHR) is transcribed at a similar level between ci and Actb-SLa:GFP in all organs examined (Fig.…”

“…Although SL may (or may not; as exemplified by the above conflicting evidence between species) have important functions during these circumstances, its direct effects on the body remain unknown from these observations. Several in-vitro/vivo experiments using purified/recombinant SL revealed its physiological activities in steroidogenesis (Planas et al, 1992), phosphate transport (Lu et al, 1995), melanosome aggregation (Zhu and Thomas, 1997), phagocyte activation (Calduch-Giner et al, 1998; but see Sakai et al, 1996), lipolysis (Vega-Rubín de Celis et al, 2003), etc. However, because the effects are often weak despite the rather high doses of SL (N100 ng/ml), it is still unclear whether or not endogenous SL (∼ 20 ng/ml in the plasma; Rand-Weaver et al 1995;Zhu and Thomas 1996;Diez et al, 2007) actually plays significant roles in these activities.…”

Signal transduction mechanisms for autocrine/paracrine regulation of somatolactin-α secretion and synthesis in carp pituitary cells by somatolactin-α and -β

“…Similar to GH and PRL, SL is a member of the class I cytokines with the typical feature of four ␣-helixes arranged in an up-up-down-down topography (16). To date, SL has been associated with diverse functions in fish models, including reproduction (29,36), stress (21), lipid metabolism (40), background adaptation (53,55), phosphate transport (25), melanosome aggregation (28,54), and immune responses (4). Although most of the above-mentioned functions attributed to SL were based on correlational studies, the functional role of SL in color presentation has been unequivocally demonstrated in fish species.…”

Medaka Receptors for Somatolactin and Growth Hormone: Phylogenetic Paradox Among Fish Growth Hormone Receptors

“…Researchers have isolated SLs from many fish species and detected up-and downregulation of its expression during variable physiological processes (see references in Fukada et al 2005). Experiments using recombinant proteins of SLs show certain biological activities in vitro and in vivo (Lu et al 1995;Calduch-Giner et al 1998;Vega-Rubin de Celis et al 2003). However, none has been supported by clear and direct evidence explaining the mechanisms or actions of target cells, tissues, and organs of SLs.…”