Influence of suckling on tubulin-dependent GTPase activity in the anterior pituitary lobe of the lactating rat

Abstract

A GTPase assay was employed to determine the relative proportions of the enzymatic activity in soluble and polymerized tubulin pools in the anterior pituitary lobe of the lactating rat. The GTPase activity in the tubulin fractions was estimated in 25-50 micrograms protein using [gamma-32P]GTP. The liberation of inorganic phosphate (Pi) was proportional to the protein concentration with either of the tubulin fractions. The enzymatic activity appeared to reach equilibrium by 1 min. Antitubulin antibodies inhibited the enzymatic activity in a concentration-dependent manner in both the tubulin fractions; at a final dilution of 1:2000 the antibody maximally inhibited the enzyme activity in both the tubulin fractions by 39-44%. After establishing the optimal conditions for the GTPase assay, the effect of suckling on pituitary GTPase activity was studied. Soluble and polymerized tubulin fractions were prepared from anterior pituitaries obtained from lactating rats killed after suckling for 30, 60, and 90 min; GTPase activity was assayed in both the tubulin fractions in the absence of antitubulin antibody. Compared to the nonsuckled control, suckling for 60 and 90 min stimulated the enzymatic activity in the soluble tubulin fraction by 80% and 44%, respectively (P less than 0.05). The enzymatic activity in the polymerized tubulin fraction increased by 30% at 60 min and decreased by about 20% at 90 min (P less than 0.05). The suckling-stimulated GTPase activity in the two pituitary fractions cannot be attributed to tubulin alone since there are other proteins also capable of hydrolyzing GTP. Therefore, GTPase activity was assayed in the pituitary tubulin fractions in the presence of antitubulin antibody (1:2000 dilution); tubulin-GTPase activity is the difference between the activity assayed in the absence of the antibody and that which was determined in the presence of the antibody. In the soluble tubulin fraction, tubulin-GTPase activity increased by 166% at 30 min suckling (P less than 0.05), decreased by 40% at 60 min (P less than 0.05), and again increased by 148% at 90 min (P less than 0.05). In the polymerized tubulin fraction, the enzyme activity decreased by 82% at 30 min (P less than 0.05), increased by 742% at 60 min (P less than 0.05), and again decreased by 95% at 90 min (P less than 0.05). Thus, an inverse relationship between tubulin-GTPase activities in the two pituitary fractions was observed and provides further evidence in support of our hypothesis that microtubules are recruited to transport PRL granules from the Golgi apparatus to the plasma membrane.