Sulfate activation and transport in mammals: system components and mechanisms.

Extensive studies on the mammalian sulfate-activating enzymes and PAPS translocase have enhanced our understanding of the overall pathway of sulfate activation and utilization. Isolation of the PAPS-synthesizing activities from rat chondrosarcoma and preparation of stable non-hydrolyzable analogs of APS and PAPS have facilitated the kinetic characterization of mammalian ATP sulfurylase and APS kinase. These studies provided the basis for further experimental work showing that APS, the labile intermediate product, is channeled directly between the sulfurylase and kinase active sites. The defect in the brachymorphic mutant mouse lies in this channeling mechanism, thus interfering with efficient PAPS production. The rat chondrosarcoma ATP sulfurylase and APS kinase activities, in fact, reside in a single bifunctional cytoplasmic protein, which has now been cloned and expressed. The mechanism by which PAPS reaches its sites of utilization in the Golgi lumen has also been elucidated: The PAPS translocase is a 230-kDa integral Golgi membrane protein which functions as an antiport.

Kinetics of PAPS translocase: evidence for an antiport mechanism.

In order to gain an understanding of the mechanisms involved in the transfer of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) from the cytosol where it is synthesized to the Golgi lumen where it serves as the universal sulfate donor for sulfate ester formation in higher organisms, we have undertaken a kinetic characterization of the PAPS translocase from rat liver Golgi. Analyzing the PAS translocase activity in both intact Golgi vesicles and in a reconstituted liposome system, we have determined a number of physical and kinetic parameters. Strong competitive inhibition in zero-trans uptake experiments only with beta-methylene PAPS and adenosine 3',5'-biphosphate (PAP) suggest the transporter is highly specific for the 3'-phosphate. The demonstration of trans acceleration as observed by stimulation of transport activity under exchange conditions suggests that the translocase is a carrier with distinct binding sites accessible from both faces of the membrane. The behavior of the PAPS translocase in the presence of equilibrium concentrations of PAP supports the function of an antiport mechanism. Thus the translocase is characterized by its kinetic properties as a specific transporter of PAPS which acts through an antiport mechanism with PAP as the returning ligand. This characterization of the transport activity has proved instrumental in the identification of an approximate 230 kDa Golgi membrane protein as the PAPS translocase protein [Ozeran, J.D., Westley, J., & Schwartz, N.B. (1996) Biochemistry 35, 3695-3703 (accompanying paper)].

Identification and partial purification of PAPS translocase.

Sulfation of all macromolecules in higher organisms requires the high-energy donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS). PAPS is synthesized via the sequential actions of two cytoplasmic enzymes, ATP sulfurylase and APS kinase, and then must be transferred across the Golgi membrane for utilization by lumenal sulfotransferases. Following the kinetic characterization of the PAPS translocase as a specific transporter that act through an antiport mechanism with PAP as the returning ligand [Ozeran, J.D., Westley, J., Schwartz, N.B. (1996) Biochemistry 35, 3685-3694 (accompanying paper)], the present study describes the identification and physical characterization of the PAPS translocase from rat liver Golgi membranes. The following evidence suggests the PAPS translocase is a membrane spanning protein of approximately 230 kDa: isolation by affinity chromatography on beta-methylene PAPS matrices of a 230 kDa Golgi membrane protein concomitant with PAPS translocase activity; demonstration that the 230 kDa protein possesses the only PAPS binding site accessible to the cytoplasmic face of intact Golgi membranes, while several other PAPS binding proteins are labeled in solubilized membrane preparations; reduction in size of the 230 kDa membrane protein and loss of PAPS translocase activity following protease treatment; estimation via hydrodynamic analysis of a molecular size of the membrane protein associated with PAPS translocase activity; and correlation of beta-methylene PAPS binding and labeling of the 230 kDa Golgi protein with PAPS translocase activity in artificial liposomes. These and the accompanying data have permitted the identification of the first of a potentially large class of Golgi membrane nucleotide-metabolite transporters.

Intermediate channeling between ATP sulfurylase and adenosine 5'-phosphosulfate kinase from rat chondrosarcoma.

Biosynthesis of the activated sulfate donor PAPS (3'-phosphoadenosine 5'-phosphosulfate) involves the sequential action of two enzyme activities. ATP sulfurylase catalyzes the formation of APS (adenosine 5'-phosphosulfate) from ATP and free sulfate, and APS is then phosphorylated by APS kinase to produce PAPS. Using rat chondrosarcoma ATP sulfurylase and APS kinase, a newly developed assay system, which permits measuring the accumulation of both APS and PAPS in the presence of both enzyme activities, produces a PAPS/APS ratio corresponding to a "channeling efficiency" of 96%. The velocity of the APS kinase reaction measured in the overall system with endogenously synthesized APS is 8-fold greater than that of the isolated kinase reaction using exogenous APS. Most conclusively, isotope dilution and enrichment experiments show that the APS intermediate does not equilibrate with APS in the bulk medium but remains largely bound in the rat enzyme system. In contrast, control experiments with a nonchanneled system containing a mixture of the sulfurylase and kinase isolated from Penicillium chrysogenum give the results expected for a nonchanneled pathway. These data indicate that APS is channeled between the active sites of ATP sulfurylase and APS kinase during the production of PAPS in rat chondrosarcoma.