Ontogeny of guanine-nucleotide-binding regulatory proteins in rabbit liver

Abstract

Ontogeny of trimeric GTP-binding regulatory proteins (G-proteins) and their subunits in rabbit liver during neonatal development was studied, by using bacterial-toxin-catalysed ADP-ribosylation of membrane proteins, immunoblot analysis to quantify the alpha-subunit (alpha s and alpha i) of stimulatory (Gs) and inhibitory (Gi) G-protein and the beta-subunit, and reconstitution assay with cyc- membranes (from Gs-deficient variant of S49 lymphoma cell) to measure Gs activity. Under optimal conditions of ADP-ribosylation, little cholera-toxin substrate (alpha s) was detected in membranes from liver of neonatal animals up to 24 h of age. Thereafter ribosylatable alpha s proteins, i.e. 45 kDa (alpha s-1) and 52 kDa (alpha s-2) proteins, were increasingly evident, reaching maximal levels in membranes from animals aged 4-6 weeks. The concentrations of alpha s-1 and alpha s-2, as determined by immunoblotting, were 6.1 +/- 0.8 and 2.7 +/- 0.4 pmol/mg of protein respectively at birth, and did not change during 0-24 h after birth. Thereafter they gradually increased to maximal levels of 22.1 +/- 1.3 and 10.5 +/- 0.7 pmol/mg of protein for alpha s-1 and alpha s-2 respectively, within 6 weeks. The beta-subunit also showed a similar 3-4-fold increase during the same age span. In contrast, the pertussis-toxin substrate (alpha i) was clearly evident even in membranes from term animals and in all age groups studied. Its developmental pattern, as assessed by ADP-ribosylation, was the same as that determined by immunoblot analysis. The functional activity of Gs in cholate extracts of membranes exhibited similar developmental pattern to that of cholera-toxin-mediated labelling. This activity also paralleled the concentrations of alpha s as measured by immunoblotting. These results suggest differential expression of G-protein subunits in liver during neonatal development.