Assay of ATP-sulfurylase activity from rat liver by high-performance liquid chromatography.

Alteration of proteoglycan composition is known to accompany morphogenesis. In many tissues one such alteration is the removal of hyaluronate and its replacement with a sulfated proteoglycan. Several mechanisms that could regulate this alteration have been studied leading to a hypothesis that the increase in the sulfated proteoglycan is regulated by controlling the activity of those enzymes involved in the activation of the sulfate. To measure any variations in these activities usually begins with a purification of the enzyme. However, this procedure is difficult to perform where tissue samples are difficult to obtain in large enough quantities. Therefore, the examination of an enzymatic activity when tissue samples are in short supply requires the development of methods for the assay of the specific activity after a minimum of purification. In this paper we report on the development of just such an assay for ATP-sulfurylase, the enzyme that catalyses the first step in the activation of sulfate. This method uses anion-exchange high-performance liquid chromatography and differs from a previously published procedure [F. A. Hommes and L. Moss, Anal. Biochem., 154 (1986) 100] in that the compounds are detected spectrophotometrically instead of radiometrically and also in that the ATP, ADP, AMP and their sulfated analogues, adenosine 5'-phosphosulfate and 3'-phosphoadenosine 5'-phosphosulfate, are separated isocratically. Studies performed with 35SO4(2-) were used to validate this new method. The separation of all these compounds has allowed us to develop a one-step, on-line assay procedure which can be performed on small samples of partially purified preparations. We have used this procedure to measure the activity of the ATP sulfurylase in extracts of rat liver and tongue. Our results indicated that the ATP-sulfurylase activity from rat liver was soluble with a pH optimum of 8.0. The identity of the reaction product was verified using radiolabeled sulfate as the substrate and recovering the radiolabel in the product. Preliminary kinetic studies with this method showed the sulfurylase activity to have an apparent Michaelis constant of 3 microM and a maximal velocity of 0.56 pmol/min per mg protein.