Selenium-isotopic signature toward mass spectrometric identification and enzyme activity assay.

The unraveling of enzymatic reactions, especially identification of enzymatic substrates or products, is important to elucidate biological processes. Here a selenium-isotopic signature for mass spectrometric identification of enzymatic-related species is demonstrated by using selenium-containing peptides (SePeps) as substrates. Thus a strategy is proposed for rapid and precise assay of multiple enzyme activity. These SePeps can be synthesized by introduction of one selenomethionine residue in the sequence and simply identified in the full-scan mode with the feature of distinctive selenium-isotopic distribution without MS/MS verifications, which proposes a novel solution to the specific identification of enzyme-related species, allows to exclude the interferences of species with tiny mass differences in bio-samples, and meanwhile can offer a judgement on data accuracy for the analysis of enzyme activities. As a proof-of-concept, a method for multiple analysis of two representative enzymes in MCF-7 cell lysate has been developed with the isotopic peak areas of either SePep substrates or enzymatic products with the top intensities. These results could be the foundation to extend the method for more complicated enzyme systems. The selenium-isotopic signature provides a powerful protocol for high-throughput assays of peptide-metabolizing enzymes with enhanced confidence and can be extended to screen enzymatic reaction-related substrates.

Activation of extracellular-signal regulated kinase by epidermal growth factor is potentiated by cAMP-elevating agents in primary cultures of adult rat hepatocytes.

We investigated the effects of α- and ß-adrenergic agonists on epidermal growth factor (EGF)-stimulated extracellular-signal regulated kinase (ERK) isoforms in primary cultures of adult rat hepatocytes. Hepatocytes were isolated and cultured with EGF (20 ng/ml) and/or α(1)-, α(2)- and ß(2)-adrenergic agonists. Phosphorylated ERK isoforms (ERK1; p44 mitogen-activated protein kinase (MAPK) and ERK2; p42 MAPK) were detected by Western blotting analysis using anti-phospho-ERK1/2 antibody. The results show that EGF induced a 2.5-fold increase in ERK2-, but not ERK1-, phosphorylation within 3 min. This EGF-induced ERK2 activation was abolished by treatment with the EGF-receptor kinase inhibitor AG1478 (10(-7) M) or the MEK (MAPK kinase) inhibitor PD98059 (10(-6) M). The α(2)-adrenergic and ß(2)-adrenergic agonists, UK14304 (10(-6) M) and metaproterenol (10(-6) M), respectively, had no effect in the absence of EGF, but metaproterenol significantly potentiated EGF-induced ERK2 phosphorylation. Moreover, the cell-permeable cAMP analog 8-bromo cAMP (10(-7) M), also potentiated EGF-induced ERK2 phosphorylation. The effects of these analogs were antagonized by the protein kinase A (PKA) inhibitor H-89 (10(-7) M). These results suggest that direct or indirect activation of PKA represents a positive regulatory mechanism for EGF stimulation of ERK2 induction.

A sexually dimorphic distribution pattern of the novel estrogen receptor G-protein-coupled receptor 30 in some brain areas of the hamster.

The isolation of the G-protein-coupled receptor 30 (GPR30), an orphan membrane receptor unrelated to nuclear estrogen receptors (ERs), has become a key factor towards the unraveling of rapid estrogen action. This membrane receptor together with cellular signaling intermediaries, i.e., extracellular signal-dependent kinases 1 and 2, may promote neuronal proliferation and differentiation activities. In the present study, an evident gene expression pattern of GPR30 characterized postnatal 7 (young) and 60 (adult) days of age hamsters as shown by its heterogeneous mRNA distribution in hypothalamic, amygdalar and cerebellar areas of both sexes. In particular, most of the brain areas considered in the adult hamster plus only the amygdala and cerebellum of young animals behaved in a sexually dimorphic fashion. This similar pattern was also detected for the ERalpha and beta, as shown by the latter receptor prevailing in young and adult females, while the former predominated in young females. Even for the two kinases, a sexually dimorphic distribution was featured above all for young hamsters. Overall, the findings of the present study established a distinct expression pattern of the novel ER (GPR30) that may operate differently in some brain areas of the hamster and this may provide interesting insights regarding its probable neuroprotective role during the execution of some hibernating states, which are typical of our rodent model.